EP1140360A1 - Dispositifs de prelevement de tissu contenant des biocapteurs - Google Patents
Dispositifs de prelevement de tissu contenant des biocapteursInfo
- Publication number
- EP1140360A1 EP1140360A1 EP99965340A EP99965340A EP1140360A1 EP 1140360 A1 EP1140360 A1 EP 1140360A1 EP 99965340 A EP99965340 A EP 99965340A EP 99965340 A EP99965340 A EP 99965340A EP 1140360 A1 EP1140360 A1 EP 1140360A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- preferred
- nucleic acid
- electrode
- probe
- attachment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2610/00—Assays involving self-assembled monolayers [SAMs]
Definitions
- the invention relates to tissue collection devices such as blood collection devices that comprise biosensors for the detection of target analytes such as nucleic acids and proteins, including antibodies and enzymes
- these tests can be classified into several categories, including nucleic acid tests (for pathogens, oncogenes, genetic diseases, etc ) and immunoassay tests (for a wide variety of antigens and antibodies)
- nucleic acid tests for pathogens, oncogenes, genetic diseases, etc
- immunoassay tests for a wide variety of antigens and antibodies
- tissue collection devices that have incorporated biosensors, particularly electrodes, for use in diagnosis and screening
- the present invention provides tissue collection devices, particularly blood collection devices, comprising an electrode
- the electrodes may further comprise self-assembled monolayers and capture binding ligands, particularly nucleic acid capture probes
- the monolayers may comprise insulators and/or electroconduit-forming species (EFS)
- EFS electroconduit-forming species
- the devices may further comprise at least one reagent, including anticoagulants, probe nucleic acids, and lysis reagents
- the invention provides method of detecting a target analyte is a sample comprising applying an initiation signal to a tissue collection device comprising an electrode
- the electrode may comprise a self-assembled monolayer and an assay complex comprising a capture binding ligand, the target analyte and an electron transfer moiety
- the methods further comp ⁇ se detecting electron transfer between the electrode and the electron transfer moiety
- the methods may further comprising collecting the sample, for example using blood collection equipment
- Figures 1A, 1 B, 1 C and 1 D depict tissue collection devices configured with reservoirs
- Figure 1A shows the tube 10 with a cap 20
- the cap 20 has a reservoir 30 with reagent(s) 40
- Figure 1 B is similar except that there are two reservoirs 30 and 31 with reagents 40 and 41
- Figure 1 C depicts a similar configuration except that the reservoir 30 is part of the tube 10 rather than the cap 20
- Figure 1 D depicts a configuration that utilizes a membrane 50 at the end of the tube It should be noted that none of the systems depicted in Figure 1 show the biosensor
- Figures 2A and 2B depict tissue collection devices with reservoirs for testing of a portion of the sample
- Figure 2A depicts a device that utilizes a cap 20 as will be appreciated by those in the art, devices with inlet ports membranes, and other configurations may be used as well
- the reservoir 30 (with optional reagents 40) is separated from the sample area 70 by a valve or seal 60
- Figure 2B depicts a similar system except that the reservoir 30 can be detached from the sample chamber 70, using an detachable union 65 It should be noted that none of the systems depicted in Figure 2 show the biosensor
- Figures 3A, 3B and 3C depict some of the possible configurations of the biosensor and external leads for the compositions of the invention
- Figure 3A depicts the electrode of the biosensor 80 in the cap 20 with interconnects 90
- Figures 3B and 3C depict the electrode of the biosensor 80 as a component of the tube 10
- Figure 3B depicts a single biosensor
- Figure 3C depicts multiple biosensors 80.
- Figure 3C depicts one set of external leads for all the electrodes, although as will be appreciated by those in the art, multiple leads may be used as well
- there may be physical barriers between compartments of the tube each with a biosensor
- Figure 3D depicts a biosensor 80 in a reservoir 30 of a device 10
- Figures 4A, 4B and 4C depict three preferred embodiments for attaching a target sequence to the electrode Although generally depicted as nucleic acids, non-nucleic acid embodiments are also useful
- Figure 4A depicts a target sequence 120 hybridized to a capture probe 100 linked via a attachment linker 106, which as outlined herein may be either a conductive oligomer or an insulator
- the capture probes may be the same, or the capture probes at any particular pad may be different, comprising sequences that hybridize to different parts of the target
- the electrode 105 comprises a monolayer of passivation agent 107, which can comprise conductive oligomers (herein depicted as 108) and/or insulators (herein depicted as 109)
- n is an integer of at least 1 , although as will be appreciated by those in the art, the system may not utilize a capture probe at all (i e n is zero), although this is generally not preferred
- the upper limit of n will depend on the length of the target sequence and the required sensitivity
- Figure 4B depicts the use of a single capture extender probe 110 with a first portion
- FIG. 4C depicts the use of two capture extender probes 110 and 130
- the first capture extender probe 110 has a first portion 111 that will hybridize to a first portion of the target sequence 120 and a second portion 112 that will hybridize to a first portion 102 of the capture probe 100
- the second capture extender probe 130 has a first portion 132 that will hybridize to a second portion of the target sequence 120 and a second portion 131 that will hybridize to a second portion 101 of the capture probe 100
- Figures 5A, 5B, 5C, 5D and 5E depict different possible configurations of label probes and attachments of ETMs
- the figures depict a second binding ligand that is a hyb ⁇ dizable nucleic acid, but as will be appreciated by those in the art, the second binding ligand can be non-nucleic acid as well
- the second binding ligand can be non-nucleic acid as well
- Figures 6A, 6B, 6C, 6D, 6E, 6F and 6G depict some of the embodiments of the invention All of the monolayers depicted herein show the presence of both conductive oligomers 108 and insulators 107 in roughly a 1 1 ratio, although as discussed herein, a variety of different ratios may be used, or either species may be completely absent, or other monolayer-forming species may be used. In addition, as will be appreciated by those in the art, any one of these structures may be repeated for a particular target sequence, that is, for long target sequences, there may be multiple assay complexes formed Additionally, any of the electrode-attachment embodiments of Figure 4 may be used in any of these systems
- Figures 6A, 6B and 6D have the target sequence 120 containing the ETMs 135, as discussed herein, these may be added enzymatically, for example during a PCR reaction using nucleotides modified with ETMs, resulting in essentially random incorporation throughout the target sequence, or added to the terminus of the target sequence
- Figure 6D depicts the use of two different capture probes 100 and 100', that hybridize to different portions of the target sequence 120 As will be appreciated by those in the art, the 5'-3' orientation of the two capture probes in this embodiment is different
- Figure 6C depicts the use of label probes 145 that hybridize directly to the target sequence 120
- Figure 6C shows the use of a label probe 145, comprising a first portion 141 that hybridizes to a portion of the target sequence 120, a second portion 142 comprising ETMs 135
- a particularly preferred embodiment for use in tissue collection devices is the use of direct capture of the target sequence to the capture probe, with at least one label probe hybridizing to a different portion of the target
- Figures 6E, 6F and 6G depict systems utilizing label probes 145 that do not hybridize directly to the target, but rather to amplifier probes that are directly (Figure 6E) or indirectly (Figures 6F and 6G) hybridized to the target sequence
- Figure 6E utilizes an amplifier probe 150 has a first portion 151 that hybridizes to the target sequence 120 and at least one second portion 152, i e the amplifier sequence, that hybridizes to the first portion 141 of the label probe Figure 6F is similar, except that a first label extender probe 160 is used comprising a first portion 161 that hybridizes to the target sequence 120 and a second portion 162 that hybridizes to a first portion 151 of amplifier probe 150 A second portion
- the amplifier probe 150 hybridizes to a first portion 141 of the label probe 140, which also comprises a recruitment linker 142 comprising ETMs 135 Figure 6G adds a second label extender probe 170, with a first portion 171 that hybridizes to a portion of the target sequence 120 and a second portion that hybridizes to a portion of the amplifier probe
- Figure 6H depicts a system that utilizes multiple label probes The first portion 141 of the label probe
- Figures 7A, 7B, 7C, 7D, 7E and 7F depict some ' mechan ⁇ sm-1 " detection systems
- Figure 7A shows portions of target sequence 120 hyb ⁇ dized to detection probes 300 linked via conductive oligomers 108 to electrode 105
- the hybridization complex comprises an ETM 135 that can be covalently linked either to the target sequence or the detection probe 300, or non-covalently (i e a hybridization indicator)
- the monolayer is depicted with insulators 107, although as outlined herein, any type of passivation agent may be used
- Figure 7B depicts the use of capture probe 100 linked via attachment linker 106 to electrode 105, although as will be appreciated, since the label probe 145 hybridizes to the detection probe 300 this can serve as a type of capture probe
- a label probe 145 comprising a first portion 141 that hybridizes to a portion of the target sequence 120 and a second portion 142 that hybridizes to the detection probe 300
- Figure 7C depicts
- Figure 7E depicts a similar system, but uses label probes 145 comprising a first portion 141 that hybridizes to the amplification sequence 152 and a second portion 142 that hybridizes to a detection probe 300
- Figure 7F depicts the same thing but using a linear amplification probe 150
- the present invention is directed to the use of biosensors in a tissue collection device
- the ability to collect the tissue sample, such as a blood sample, in a container that comprises a biosensor (and optionally assay reagents) allows a one-step molecular diagnostic assay
- a tissue sample such as a blood sample
- a container that comprises a biosensor and optionally assay reagents
- the donation of blood into a container that has a biosensor allows the rapid and inexpensive testing of blood for contaminants, including pathogens such as viruses (hepatitis strains, HIV, etc ) or bacteria with a minimum of blood handling
- tissues collected specifically for the purpose of testing such as blood or vaginal samples for sexually transmitted diseases, can also be tested rapidly with a minimum of handling
- tissue collection devices By “tissue” or grammatical equivalents herein is meant any tissue for which a bioassay can be done, including, but not limited to, blood, lymph, saliva, vaginal and anal secretions, urine, feces, perspiration, tears, and other bodily fluids
- tissue collection device herein is meant any container, generally capable of being sealed, that can be used to collect, contain or store tissues, including bodily fluids
- Tissue collection devices are generally sterile Specifically included herein are blood collection devices, such as are well known in the art, including blood collection tubes and bags, including VACUTAINER® brand evacuated tubes
- the tissue collection devices of the invention can be configured in a variety of ways
- the device has a single compartment for receiving sample, that may include optional reagents prior to collection
- the tissue collection device is configured to have more than one compartment This may be done in a variety of ways, depending on the use of the device For example, when the entire sample is to be used for assay, the compartments may be used for storage of assay reagents Alternatively, when only a portion of the collected sample is to be assayed, the compartments may be used to segregate the portion for subsequent assay, or both
- the entire sample is used in the assay
- U S Patent No 6,001 ,087 describes a collection assembly wherein the cap of the device comprises a reservoir containing reagents as defined below The insertion of a needle, such as is done during blood drawing, results in the rupture of the reservoir and the addition of the reagents to the sample This is generally shown in Figures 1A and 1 B, showing one and multiple compartments for rupture in this manner.
- the reservoir may be part of the tube, rather than the cap, as is generally depicted in Figure 1 C
- the reservoir may be at the end of the tube, and utilize a membrane that can rupture upon centrifugation, as is shown in Figure 1 D
- a gel barrier may be used, as is used in VACUTAINER® PPTTM systems
- the device can have a compartment to allow the testing of only a portion of the sample
- the tissue collection tube comprises reagents
- reagents used in tissue handling, such as anticoagulants for blood, as well as a variety of other reagents including salts, buffers, neutral proteins, e g albumin, detergents, etc which may be used to facilitate optimal binding of the target analyte to the sensor composition and detection, and/or reduce nonspecific or background interactions
- reagents that otherwise improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc , may be used, depending on the sample preparation methods and purity of the target
- there may be reagents and other components used to accelerate the binding of the target analytes to the biosensors such as are generally described in PCT US99/14191 , hereby incorporated by reference in its entirety #
- reagents comprising components of the bioassay, including for example amplification probes, label probes, capture extender probes, etc , as is generally outlined in U S Patent Nos 5,591 ,578, 5,824,473, 5,770,369, 5,705,348, 5,780,234, and 5,952,172, WO98/20162, W099/37819, PCT US98/12430, PCT US99/01703, PCT US98/12082, PCT/US99/14191 , PCT/US99/21683,
- PCT/US99/25464, and PCT US99/10104, and U S S N s 09/135,183, 09/295,691 and 09/306,653, can be placed in the tube prior to, after or during the introduction of the sample tissue
- the reagent is a cell lysis agent for the lysis of cells (including patient cells, bacteria, etc ) or other pathogens such as virus
- Suitable lysis buffers include, but are not limited to, guanidium chloride, chaotropic salts, enzymes such as lysozymes, etc
- a simple dilution with water or buffer can result in hypotonic lysis
- the lysis agent may be in solution form or solid form that is taken up in solution upon introduction of the sample
- one or more reservoirs of the tissue collection device comprises the reagents
- the tissue collection device further comprises a biosensor as outlined herein
- the biosensor may comprise a single substrate with a single electrode, multiple substrates each with one or more electrodes, or a single substrate with a plurality of electrodes in an array format
- the placement of the biosensor in the device can be done in a variety of ways, as is generally depicted in Figure 3
- the biosensor is in the cap or stopper of the device as shown in Figure 3A
- there are leads or interconnects on the outside of the cap with the biosensor contacting the sample on the inside of the device
- what is important is that the biosensor have some contact with some or all of the sample to be assayed
- the placement of the b ⁇ osensor(s) is on a surface internal to the sample compartment, as is generally depicted in Figures 3B and 3C
- the external leads may be on the sample compartment or on the cap
- different biosensors may be located in different sections of the device For example, detection of genomic sequences may be done in one section of the tube, for example near the end, where the heavier blood cells are after centrifugation, and detection of pathogenic (particularly viral) sequences may be done in a different section, for example where the plasma resides after centrifugation
- biosensors may be located in different physical compartments of the device; for example, the use of gel barriers or other membranes may allow certain cell types to be compartmentalized in the sample surface, thus reducing the complexity of the sample in any one compartment
- biosensors a sensor capable of detecting the presence of a target analyte in a sample.
- Biosensors in this context includes electrochemical sensors, optical fiber sensors, evanescent wave sensors, surface plasmon resonance sensors and acoustic wave sensors
- target analyte or “analyte” or grammatical equivalents herein is meant any molecule or compound to be detected and that can bind to a binding species, defined below Suitable analytes include, but not limited to, small chemical molecules such as environmental or clinical chemical or pollutant or biomolecule, including, but not limited to, pesticides, insecticides, toxins, therapeutic and abused drugs, hormones, antibiotics, antibodies, organic materials, etc Suitable biomolecules include, but are not limited to, proteins (including enzymes, immunoglobulins and glycoproteins), nucleic acids, lipids, lectms, carbohydrates, hormones, whole cells (including procaryotic (such as pathogenic bacteria) and eucaryotic cells, including mammalian tumor cells), viruses, spores, etc Particularly preferred analytes are proteins including enzymes, drugs, cells, antibodies, antigens, cellular membrane antigens and receptors (neural, hormonal
- analytes may be detected using the present methods basically any target analyte for which a binding ligand, described below, may be made may be detected using the methods of the invention
- nucleic acid or “ohgonucleotide” or grammatical equivalents herein means at least two nucleotides covalently linked together
- a nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below nucleic acid analogs are included that may have alternate backbones, comprising, for example phosphoramide (Beaucage et al , Tetrahedron 49(10) 1925 (1993) and references therein, Letsmger, J Org Chem 35 3800 (1970), Sp ⁇ nzl et al , Eur J Biochem 81 579 (1977), Letsinger et al , Nucl Acids Res 14 3487 (1986), Sawai et al, Chem Lett 805 (1984), Letsinger et al , J Am Chem Soc 1 10 4470 (1988), and Pauwels et al , Chemica Sc ⁇ pt
- nucleic acid analogs may find use in the present invention
- mixtures of naturally occurring nucleic acids and analogs can be made, for example at the site of surface species or ETM attachment, an analog structure may be used
- mixtures of different nucleic acid analogs, and mixtures of naturally occunng nucleic acids and analogs may be made
- PNA peptide nucleic acids
- These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids
- Tm melting temperature
- RNA typically exhibit a 2-4 ° C drop in Tm for an internal mismatch
- the drop is closer to 7-9 ° C
- hybridization of the bases attached to these backbones is relatively insensitive to salt concentration This is particularly advantageous in the systems of the present invention, as a reduced salt hybridization solution has a lower Faradaic current than a physiological salt solution (in the range of 150 mM)
- the nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence
- the nucleic acid may be DNA, both genomic and cDNA,
- RNA or a hybrid where the nucleic acid contains any combination of deoxy ⁇ bo- and ⁇ bo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosme, guanine, inosine, xathanine hypoxathanme, isocytos e, isoguanine, etc
- a preferred embodiment utilizes isocytosine and isoguanine in nucleic acids designed to be complementary to other probes, rather than target sequences, as this reduces non-specific hybridization, as is generally described in U S Patent No
- nucleoside includes nucleotides as well as nucleoside and nucleotide analogs, and modified nucleosides such as ammo modified nucleosides
- nucleoside' includes non-naturally occunng analog structures
- the target analyte is a target sequence
- target sequence or “target nucleic acid” or grammatical equivalents herein means a nucleic acid sequence on a single strand of nucleic acid
- the target sequence may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA and rRNA, or others
- the target sequence may be a target sequence from a sample, or a secondary target such as a product of an amplification reaction, etc It may be any length, with the understanding that longer sequences are more specific
- the complementary target sequence may take many forms For example, it may be contained within a larger nucleic acid sequence, i e all or part of a gene or mRNA, a restriction fragment of a plasmid or genomic DNA, among others
- probes are made to hybridize to target sequences to determine the presence or absence of the target sequence in a
- the methods of the invention are used to detect pathogens such as bacteria
- preferred target sequences include rRNA, as is generally described in U S Patent Nos 4,851 ,330, 5,288,611 , 5,723,597, 6,641 ,632, 5,738,987, 5,830,654, 5,763,163,
- compositions of the invention comprising biosensors with assay complexes in tissue collection devices can be used in a number of standard diagnostic te ⁇ ts
- These assays include, but are not limited to, detection of Adenosme Deaminase Gene, Adenovirus, Adrenoleukodystrophy Gene, Adult Polycystic Kidney Disease, Aeromonas spp.; Pleistomonas spp , Alpha,-Ant ⁇ tryps ⁇ n Deficiency, ⁇ - Thalassemia Detection, Alzheimer's Disease,
- Amebae, Androgen Receptor Gene Antimicrobial Resistance Determination, Arylsulfatase A Gene, Aspartoacylase Gene, ⁇ -thalassemia, Babesta mtcroti, Bartonella, Basal Cell Carcinoma; bcl-2 Gene Rearrangements, bcr Gene Rearrangements, Bordetella spp , Borrelia burgdodert, B/T Cell Gene Rearrangements, c-myc, c-src Proto-Oncogene, Campylobacter spp , Arcobacter spp , Helicobacter spp , Candida albtcans, Carbonic Anhydrase II Gene, Carcinoembryonic Antigen, Charcot-Ma ⁇ e-Tooth
- Herpesv ⁇ rus-6 Types A and B Human Papillomavirus, Huntmgton's Disease Genes, Hurler/Scheie Syndrome Gene, Insulin Gene, Insulin Receptor Gene, Lammm/Laminm Receptor, Leber's Hereditary Optic Nueropathy Genes, Legionella pneumophila, Leishmania spp , Leukemia/Lymphoma Molecular Prognostic Markers, Low Density Lipoprotein Receptor and Ligands Apolipoprotems B and E, Lutemizmg Hormone Gene Medium-Chain Acyl-Coenzyme A Dehydrogenase Gene, Minimal Residual Disease Detection Mitrochond ⁇ al ATPase 6 Gene, Mitochond ⁇ al DNA, Mitrochond ⁇ al DNA Deletions, Mitrochondnal DNA Depletions Mitochond ⁇ al tRNA Genes, Molecular Epidemiology of Nosocomial
- Multi-Drug Resistance Genes Multiple Endocrine Neoplasia Type 1 , Multiple Endocrine Neoplasia Type 2A and 2B Mycobactenum leprae, Mycobacterium spp , Mycoplasma spp, Myotonic Dystrophy Gene Neissena gonorrhoeae N-myc Gene Amplification, Ornithme Transcarbamylase Gene, p53 Tumor-Suppressor Gene, Parvovirus B 19, Paternity Testing, Plasmodium spp, Porphobilinogen Deammase Gene Porphobilinogen Synthase Gene, Post-Transfusion Graft Versus
- Host Disease Protein C Gene Pyruvate Dehydrogenase Gene, Rabies Virus, ras Family of Oncogenes, Respiratory Syncytial Virus, Retinoblastoma Gene, Retmoic Acid Receptor Gene, RhD Genotypmg, Rotavirus, Rubella Virus, Schistosoma spp, Sickle Cell Disease, Thyrotropm Receptor Gene Toxoplasma gondn, Treponema pallidum, Trichomonadidae, Family, Trypanosoma spp, Tumor Metastasis Suppressor Gene - NM23, Uroporphy ⁇ nogen III Synthase Gene, Varicella-Zoster Virus, von
- any target analyte for which a binding ligand, described below, may be made may be detected using the methods of the invention While many of the techniques described below exemplify nucleic acids as the target analyte, those of skill in the art will recognize that other target analytes can be detected using the same systems
- the present invention is directed to the detection of target analytes, preferably using electrodes, in tissue collection devices
- detection of an ETM is based on electron transfer through the stacked ⁇ -orbitals of double stranded nucleic acid This basic mechanism is described in U S Patent Nos 5,591 ,578,
- the target analyte is a nucleic acid
- a non-nucleic acid target analyte is used, with an optional capture binding ligand (to attach the target analyte to the detection electrode) and a soluble binding ligand that carries a nucleic acid "tail", that can then bind either directly or indirectly to a detection probe on the surface to effect detection
- the ETM can be detected, not necessarily via electron transfer through nucleic acid, but rather can be directly detected on the surface of a monolayer That is, the electrons from the ETMs need not travel through the stacked ⁇ orbitals in order to generate a signal
- the detection electrode preferably comprises a self-assembled monolayer (SAM) that serves to shield the electrode from redox-active species in the sample
- SAM self-assembled monolayer
- the presence of ETMs on the surface of a SAM that has been formulated to comprise slight "defects” (sometimes referred to herein as "microconduits", “nanoconduits” or “electroconduits") can be directly detected
- This basic idea is termed "mechan ⁇ sm-2" herein
- the electroconduits allow particular ETMs access to the surface Without being bound by theory, it should be noted that the configuration of the electroconduit depends in part on the ETM chosen For example, the use of relatively hydrophobic ETMs allows the use of hydrophobic electrocon
- the electroconduits can be generated in several general ways, including but not limited to the use of rough electrode surfaces, such as gold electrodes formulated on PC circuit boards, or the inclusion of at least two different species in the monolayer, i e using a "mixed monolayer", at least one of which is a electroconduit-forming species (EFS)
- ETM electroconduit-forming species
- the SAMs of the invention can be generated in a number of ways and comprise a number of different components, depending on the electrode surface and the system used
- preferred embodiments utilize two monolayer forming species a monolayer forming species (including insulators or conductive oligomers) and a conductive oligomer species comprising the capture binding ligand, although as will be appreciated by those in the art, additional monolayer forming species can be included as well
- the composition of the SAM depends on the detection electrode surface
- detection electrodes comprising ' smooth” surfaces such as gold ball electrodes
- those comprising "rough” surfaces such as those that are made using commercial processes on PC circuit boards
- it appears that monolayers made on imperfect surfaces, i e rough" surfaces spontaneously form monolayers containing enough electroconduits even in the absence of exogeneous EFS, probably due
- electroconduits do not result in direct contact of sample components with the electrode surface, that is, the electroconduits are not large pores or holes that allow physical access to the electrode Rather, without being bound by theory, it appears that the electroconduits allow certain types of ETMs, particularly hydrophobic ETMs, to penetrate sufficiently into the monolayer to allow detection
- other types of redox active species including some hydrophilic species, do not penentrate into the monolayer, even with electroconduits present
- redox active species that may be present in the sample do not give substantial signals as a result of the electroconduits
- the test for a suitable SAM to reduce non-specific binding that also has sufficient electroconduits for ETM detection is to add either ferrocene or ferrocyanide to the SAM, the former should give a signal and the latter should not
- the monolayer comprises a first species comprising a conductive oligomer comprising the capture binding ligand, as is more fully outlined below, and a second species comprising a monolayer forming species, including either or both insulators or conductive oligomers
- Mechan ⁇ sm-2 system monolayers comprise a first species comprising an attachment linker comprising the capture binding ligand, a second species comprising an electroconduit-forming species (EFS), and an optional third species comprising insulators
- EDS electroconduit-forming species
- two component systems are desirable a first species comprising an attachment linker comprising the capture binding ligand and a second species comprising insulators
- the biosensors of the invention preferably comprise electrodes
- electrode herein is meant a composition, which, when connected to an electronic device, is able to sense a current or a potential and convert it to a signal Alternatively an electrode can be defined as a composition which can apply a potential to
- the electrodes described herein are depicted as a flat surface, which is only one of the possible conformations of the electrode and is for schematic purposes only
- the conformation of the electrode will vary with the detection method used
- flat planar electrodes may be preferred for optical detection methods, or when arrays of nucleic acids are made, thus requiring addressable locations for both synthesis and detection
- the electrode may be in the form of a tube, with the SAMs bound to the inner surface
- Electrode coils or mesh may e preferred in some embodiments as well This allows a maximum of surface area containing the target analyte to be exposed to a small volume of sample
- the detection electrodes are formed on a substrate
- the discussion herein is generally directed to the formation of gold electrodes, but as will be appreciated by those in the art, other electrodes can be used as well
- the substrate can comprise a wide variety of materials, as will be appreciated by those in the art, with printed circuit board (PCB) materials being particularly preferred
- PCB printed circuit board
- the suitable substrates include, but are not limited to, fiberglass, teflon, ceramics, glass, silicon, mica, plastic (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polycarbonate, polyurethanes, TeflonTM , and derivatives thereof, etc ), GETEK (a blend of polypropylene oxide and fiberglass), etc
- preferred substrate materials include printed circuit board materials
- Circuit board materials are those that comprise an insulating substrate that is coated with a conducting layer and processed using lithography techniques, particularly photolithography techniques, to form the patterns of electrodes and interconnects (sometimes referred to in the art as interconnections or leads)
- the insulating substrate is generally, but not always, a polymer
- one or a plurality of layers may be used, to make either ' two dimensional" (e g all electrodes and interconnections in a plane) or "three dimensional” (wherein the electrodes are on one surface and the interconnects may go through the board to the other side) boards
- Three dimensional systems frequently rely on the use of drilling or etching, followed by electroplating with a metal such as copper, such that the "through board” interconnections are made
- Circuit board materials are often provided with a foil already attached to the substrate, such as a copper foil, with additional copper added as needed (for example for interconnections), for example by electroplating The copper surface may then need
- the present invention provides biosensors or biochips
- each electrode preferably comprises a self-assembled monolayer as outlined herein
- one of the monolayer-forming species comprises a capture ligand as outlined herein
- each electrode has an interconnection, that is attached to the electrode at one end and is ultimately attached to a device that can control the electrode That is, each electrode is independently addressable
- the substrates are generally a component of the tissue collection device, and can be included as part of a cap or tube, or as part of a reservoir that exposes a given volume of sample to the detection electrode Generally, the detection chamber ranges from about 1 nL to 10 ml As will be appreciated by those in the art, depending on the experimental conditions and assay, smaller or larger volumes may be used
- all or part of the tissue collection device is configured as a "cartridge" that is placed into a device comprising electronic components (an AC/DC voltage source, an ammeter, a processor, a read-out display, temperature controller, light source, etc )
- electronic components an AC/DC voltage source, an ammeter, a processor, a read-out display, temperature controller, light source, etc .
- the interconnections from each electrode are positioned such that upon insertion of the cartridge into the device, connections between the electrodes and the electronic components are established
- Detection electrodes on circuit board material are generally prepared in a wide variety of ways
- high purity gold is used, and it may be deposited on a surface via vacuum deposition processes (sputtering and evaporation) or solution deposition (electroplating or electroless processes)
- the substrate must initially comprise a conductive material, fiberglass circuit boards are frequently provided with copper foil Frequently, depending on the substrate, an adhesion layer between the substrate and the gold in order to insure good mechanical stability is used
- preferred embodiments utilize a deposition layer of an adhesion metal such as chromium, titanium, titanium/tungsten, tantalum, nickel or palladium, which can be deposited as above for the gold
- electroplated metal either the adhesion metal or the electrode metal
- gram refining additives frequently referred to in the trade as b ⁇ ghteners, can optionally be added to alter surface deposition properties
- Preferred b ⁇ ghteners are mixtures of organic and inorganic acid
- the present invention provides methods of making a substrate comprising a plurality of gold electrodes
- the methods first comprise coating an adhesion metal, such as nickel or palladium
- Electroplating is preferred
- the electrode metal preferably gold, is then coated (again, with electroplating preferred) onto the adhesion metal
- the patterns of the device, comprising the electrodes and their associated interconnections are made using lithographic techniques, particularly photolithographic techniques as are known in the art, and wet chemical etching Frequently, a non-conductive chemically resistive insulating material such as solder mask or plastic is laid down using these photolithographic techniques, leaving only the electrodes and a connection point to the leads exposed, the leads themselves are generally coated
- drop deposition techniques are used to add the required chemistry, i e the monolayer forming species, one of which is preferably a capture ligand comprising species Drop deposition techniques are well known for making
- spot arrays This is done to add a different composition to each electrode, i e to make an array comprising different capture ligands
- the SAM species may be identical for each electrode, and this may be accomplished using a drop deposition technique or the immersion of the entire substrate or a surface of the substrate into the solution
- the present system finds particular utility in array formats, i e wherein there is a matrix of addressable detection electrodes (herein generally referred to "pads", “addresses” or “micro-locations”) That is, the biosensors can be made in an array format
- array herein is meant a plurality of micro-locationsi in an array format, the size of the array will depend on the composition and end use of the array Arrays containing from about 2 different capture ligands to many thousands can be made Generally, the array will comprise from two to as many as 100,000 or more, depending on the size of the electrodes, as well as the end use of the array Preferred ranges are from about 2 to about 10,000, with from about 5 to about 1000 being preferred, and from about 10 to about 100 being particularly preferred
- the compositions of the invention may not be in array format, that is, for some embodiments, compositions comprising a single capture ligand may be made as well
- multiple substrates may be used, either of different or identical
- the tissue collection devices of the invention comprise electrodes
- the electrodes of the biosensors comprise a self-assembled monolayer (SAM)
- SAM self-assembled monolayer
- ' monolayer or “self-assembled monolayer” or “SAM' herein is meant a relatively ordered assembly of molecules spontaneously chemisorbed on a surface, in which the molecules have a preferred orientation relative to each other (e g are oriented approximately parallel to each other) and a preferred orientation relative to the surface (e g roughly perpendicular to it)
- Each of the molecules includes a functional group that adheres to the surface, and a portion that interacts with neighboring molecules in the monolayer to form the relatively ordered array
- a "mixed ' monolayer comprises a heterogeneous monolayer, that is, where at least two different molecules make up the monolayer
- the SAM may comprise a variety of species including, but not limited to, EFS such as conductive oligomers, insulators, attachment linkers, etc (collectively "surface species” or ' monolayer-forming species”) In general, all
- this layer helps to prevent electrical contact between the electrodes and the ETMs, or between the electrode and extraneous electroactive species within the solvent Such contact can result in a direct "short circuit” or an indirect short circuit via charged species which may be present in the sample
- the monolayer is preferably tightly packed in a uniform layer on the electrode surface, such that a minimum of "holes" exist In this embodiment, the monolayer thus serves as a physical barrier to block solvent accesibi ty to the electrode
- the monolayer comprises electroconduit-forming species
- electroconduit-forming species or “EFS” herein is meant a molecule that is capable of generating sufficient electroconduits in a monolayer, generally of insulators such as alkyl groups, to allow detection of ETMs at the surface
- EFS have one or more of the following qualities, they may be relatively rigid molecules, for example as compared to an alkyl chain, they may attach to the electrode surface with a geometry different from the other monolayer forming species (for example, alkyl chains attached to gold surfaces with thiol groups are thought to attach at roughly 45° angles, and phenyl- acetylene chains attached to gold via thiols are thought to go down at 90° angles), they may have a structure that ste ⁇ cally interferes or interrupts the formation of a tightly packed monolayer, for example through the inclusion of branching groups such as alkyl groups, or the inclusion of highly flexible species, such as polyethylene glycol units, or they may
- EFS include conductive oligomers, as defined below, and phenyl-acetylene-polyethylene glycol species
- the EFS is not a conductive oligomer
- the EFS comprise conductive oligomers
- conductive oligomer herein is meant a substantially conducting oligomer, preferably linear, some embodiments of which are referred to in the literature as ' molecular wires'
- substantially conducting herein is meant that the oligomer is capable of transfe ⁇ ng electrons at 100 Hz
- the conductive oligomer has substantially overlapping ⁇ -orbitals, i e conjugated ⁇ -orbitals, as between the monomenc units of the conductive oligomer, although the conductive oligomer may also contain one or more sigma ( ⁇ ) bonds
- a conductive oligomer may be defined functionally by its ability to inject or receive electrons into or from an associated ETM
- the conductive oligomer is more conductive than the insulators as defined herein
- the conductive oligomers of the invention are woundto be distinguished from electroactive polymers, that themselves may donate or accept electrons.
- the conductive oligomers have a conductivity, S, of from between about 10 6 to about 10" ⁇ 1 cm 1 , with from about 10 5 to about 10 3 ⁇ 'cm 1 being preferred, with these S values being calculated for molecules ranging from about 20A to about 200A
- insulators have a conductivity S of about 10 7 ⁇ 'cm 1 or lower, with less than about 10 8 ⁇ 'cm 1 being preferred
- Desired characteristics of a conductive oligomer include high conductivity, sufficient solubility in organic solvents and/or water for synthesis and use of the compositions of the invention, and preferably chemical resistance to reactions that occur i) during binding ligand synthesis (i e nucleic acid synthesis, such that nucleosides containing the conductive oligomers may be added to a nucleic acid synthesizer during the synthesis of the compositions of the invention, n) during the attachment of the conductive oligomer to an electrode, or in) during binding assays
- conductive oligomers that will promote the formation of self-assembled monolayers are preferred
- oligomers of the invention comprise at least two monomenc subunits, as described herein As is described more fully below, oligomers include homo- and hetero-oligomers, and include polymers
- the conductive oligomer has the structure depicted in Structure 1 Structure 1
- the conductive oligomer of Structure 1 may be attached to ETMs, such as electrodes, transition metal complexes, organic ETMs, and metallocenes, and to binding ligands such as nucleic acids, or to several of these Unless otherwise noted, the conductive oligomers depicted herein will be attached at the left side to an electrode, that is, as depicted in Structure 1 , the left "Y" is connected to the electrode as described herein If the conductive oligomer is to be attached to a binding ligand, the right "Y", if present, is attached to the binding ligand such as a nucleic acid, either directly or through the use of a linker, as is described herein
- Y is an aromatic group
- n is an integer from 1 to 50
- g is either 1 or zero
- e is an integer from zero to 10
- m is zero or 1
- D is preferably carbonyl, or a heteroatom moiety, wherein the heteroatom is selected from oxygen, sulfur, nitrogen, silicon or phosphorus
- suitable heteroatom moieties include, but are not limited to, -NH and -NR
- aromatic group or grammatical equivalents herein is meant an aromatic monocyclic or polycychc hydrocarbon moiety generally containing 5 to 14 carbon atoms (although larger polycychc rings structures may be made) and any carbocyhc ketone or thioketone derivative thereof, wherein the carbon atom with the free valence is a member of an aromatic ring
- Aromatic groups include arylene groups and aromatic groups with more than two atoms removed.
- heterocycle or “heteroaryl” means an aromatic group wherein 1 to 5 of the indicated carbon atoms are replaced by a heteroatom chosen from nitrogen, oxygen, sulfur, phosphorus, boron and silicon wherein the atom with the free valence is a member of an aromatic ring, and any heterocyclic ketone and thioketone derivative thereof
- heterocycle includes thienyl, furyl, pyrrolyl, pyrimidinyl, oxalyl, mdolyl, pu ⁇ nyl, qumolyl, isoquinolyl, thiazolyl, imidozyl, etc
- the Y aromatic groups of the conductive oligomer may be different, i e the conductive oligomer may be a heterooligomer That is, a conductive oligomer may comprise a oligomer of a single type of Y groups, or of multiple types of Y groups
- the aromatic group may be substituted with a substitution group, generally depicted herein as R R groups may be added as necessary to affect the packing of the conductive oligomers, i e R groups may be used to alter the association of the oligomers in the monolayer R groups may also be added to 1 ) alter the solubility of the oligomer or of compositions containing the oligomers, 2) alter the conjugation or electrochemical potential of the system, and 3) alter the charge or characteristics at the surface of the monolayer
- R groups are preferred to increase solubility when solution synthesis is done
- the R groups, and their positions are chosen to minimally effect the packing of the conductive oligomers on a surface, particularly within a monolayer, as described below
- the R groups are chosen to minimally effect the packing of the conductive oligomers on a surface, particularly within a monolayer, as described below
- only small R groups are used within the monolayer, with larger R groups generally above the surface of the monolayer
- attachment of methyl groups to the portion of the conductive oligomer within the monolayer to increase solubility is preferred, with attachment of longer alkoxy groups, for example, C3 to C10, is preferably done above the monolayer surface
- this generally means that attachment of ste ⁇ cally significant R groups is not done on any of the first two or three oligomer subunits, depending on the average length of the molecules making up the monojayer
- R groups include, but are not limited to, hydrogen, alkyl, alcohol, aromatic, ammo, amido, nitro, ethers, esters, aldehydes, sulfonyl, silicon moieties, halogens, sulfur containing moieties, phosphorus containing moieties, and ethylene glycols
- R is hydrogen when the position is unsubstituted It should be noted that some positions may allow two substitution groups, R and R', in which case the R and R' groups may be either the same or different
- alkyl group or grammatical equivalents herein is meant a straight or branched chain alkyl group, with straight chain alkyl groups being preferred If branched, it may be branched at one or more positions, and unless specified, at any position
- the alkyl group may range from about 1 to about 30 carbon atoms (C1 -C30), with a preferred embodiment utilizing from about 1 to about 20 carbon atoms
- alkyl group (C1 -C20), with about C1 through about C12 to about C15 being preferred, and C1 to C5 being particularly preferred, although in some embodiments the alkyl group may be much larger.
- cycloalkyl groups such as C5 and C6 rings, and heterocyclic rings with nitrogen, oxygen, sulfur or phosphorus
- Alkyl also includes heteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen, and si cone being preferred
- Alkyl includes substituted alkyl groups
- substituted alkyl group herein is meant an alkyl group further comprising one or more substitution moieties "R", as defined above
- sulfur containing moieties herein is meant compounds containing sulfur atoms, including but not limited to, thia-, thio- and sulfo- compounds, thiols (-SH and -SR), and sulfides (-RSR-)
- phosphorus containing moieties herein is meant compounds containing phosphorus, including, but not limited to, phosphmes and phosphates
- silicon containing moieties herein is meant compounds containing silicon
- ether herein is meant an -O-R group
- Preferred ethers include alkoxy groups, with -0-(CH 2 ) 2 CH 3 and -0-(CH 2 ) 4 CH 3 being preferred
- ester herein is meant a -COOR group
- halogen herein is meant bromine, iodine, chlorine, or fluorine
- Preferred substituted alkyls are partially or fully halogenated alkyls such as CF 3 , etc
- aldehyde herein is meant -RCHO groups
- alcohol herein is meant -OH groups, and alkyl alcohols -ROH
- ethylene glycol or "(poly)ethylene glycol” herein is meant a -(0-CH 2 -CH 2 ) n - group, although each carbon atom of the ethylene group may also be singly or doubly substituted, i.e -(0-CR 2 -CR 2 ) n -, with R as described above
- Ethylene glycol derivatives with other heteroatoms in place of oxygen i e -(N- CH 2 -CH 2 ) n - or -(S-CH 2 -CH 2 ) n -, or with substitution groups) are also preferred
- substitution groups include, but are not limited to, methyl, ethyl, propyl, alkoxy groups such as -0-(CH 2 ) 2 CH 3 and -0-(CH 2 ) 4 CH 3 and ethylene glycol and derivatives thereof
- Preferred aromatic groups include, but are not limited to, phenyl, naphthyl, naphthalene, anthracene, phenanthro ne, pyrole, pyridine, thiophene, porphy ⁇ ns, and substituted derivatives of each of these, included fused ring derivatives
- B-D is a bond linking two atoms or chemical moieties
- B-D is a conjugated bond, containing overlapping or conjugated ⁇ -orbitals
- B-D bonds are acetylene, alkene, amide, and substituted derivatives of these three, and azo Especially preferred B-D bonds are acetylene, alkene and amide
- B or D may include carbon, nitrogen or silicon
- substitution groups are as defined as above for R
- e is preferably 1 and the D moiety may be carbonyl or a heteroatom moiety as defined above
- the terminal B-D bond may be an amide bond
- the rest of the B-D bonds may be acetylene bonds
- amide bonds when amide bonds are present, as few amide bonds as possible are preferable, but in some embodiments all the B-D bonds are amide bonds
- one type of B-D bond may be present in the conductive oligomer within a monolayer as described below, and another type above the monolayer level, for example to give greater flexibility for nucleic acid hybridization when the nucleic acid is attached via a conductive oligomer
- n is an integer from 1 to 50, although longer oligomers may also be used (see for example Schumm et al , Angew Chem Int Ed Engl 1994 33(13) 1360) Without being bound by theory, it appears that for efficient hybridization of nucleic acids on a surface, the hybridization should occur at a distance from the surface, i e the kinetics of hybridization increase as a function of the distance from the surface, particularly for long ohgonucleotides of 200 to 300 basepairs
- the length of the conductive oligomer is such that the closest nucleotide of the nucleic acid is positioned from about 6A to about 10 ⁇ A (although distances of up to 500A may be used) from the electrode surface, with from about 1 ⁇ A to about 6 ⁇ A being preferred and from about 2 ⁇ A to about 6 ⁇ A also being preferred
- n will depend on the size of the aromatic group, but generally will be from about 1 to about 20, with from about 2 to about 15 being preferred and from about 3 to about 10 being especially preferred
- m is either 0 or 1 That is, when m is 0, the conductive oligomer may terminate in the B-D bond or D moiety, i e the D atom is attached to the nucleic acid either directly or via a linker
- the conductive oligomer may terminate in the B-D bond or D moiety, i e the D atom is attached to the nucleic acid either directly or via a linker
- the conductive oligomer may terminate in the B-D bond or D moiety, i e the D atom is attached to the nucleic acid either directly or via a linker
- the D atom may be the nitrogen atom of the ammo-modified ⁇ bose
- the conductive oligomer may terminate in Y, an aromatic group, i e the aromatic group is attached to the nucleic acid or linker
- conductive oligomers falling within the Structure 1 and Structure 8 formulas, as well as other conductive oligomers, as are generally known in the art, including for example, compounds comprising fused aromatic rings or Teflon®-hke oligomers, such as -(CF 2 ) n -, -(CHF) n - and - (CFR) n - See for example, Schumm et al , Angew Chem Intl Ed Engl 33 1361 (1994), Grosshenny et al , Platinum Metals Rev 40(1 ) 26-35 (1996), Tour, Chem Rev 96 537-553 (1996), Hsung et al , Organometallics 14 4808-4815 (1995, and references cited therein, all of which are expressly incorporated by reference
- Structure 2 is Structure 1 when g is 1 Preferred embodiments of Structure 2 include e is zero, Y is pyrole or substituted pyrole, e is zero, Y is thiophene or substituted thiophene, e is zero, Y is furan or substituted furan, e is zero, Y is phenyl or substituted phenyl, e is zero, Y is pyridine or substituted pyridine, e is 1 , B-D is acetylene and Y is phenyl or substituted phenyl (see Structure 4 below)
- a preferred embodiment of Structure 2 is also when e is one, depicted as Structure 3 below
- Preferred embodiments of Structure 3 are Y is phenyl or substituted phenyl and B-D is azo, Y is phenyl or substituted phenyl and B-D is acetylene, Y is phenyl or substituted phenyl and B-D is alkene, Y is pyridine or substituted pyridine and B-D is acetylene, Y is thiophene or substituted thiophene and B-D is acetylene, Y is furan or substituted furan and B-D is acetylene, Y is thiophene or furan (or substituted thiophene or furan) and B-D are alternating alkene and acetylene bonds
- any Structure 3 oligomers may be substituted with any of the other structures depicted herein, i e Structure 1 or 8 oligomer, or other conducting oligomer, and the use of such Structure 3 depiction is not meant to limit the scope of the invention
- Particularly preferred embodiments of Structure 4 include n is two, m is one, and R is hydrogen, n is three, m is zero, and R is hydrogen, and the use of R groups to increase solubility
- the conductive oligomers are pseudopeptide oligomers
- the amide bond in Structure 5 is depicted with the carbonyl to the left, i e -CONH- , the reverse may also be used, i e -NHCO-
- Particularly preferred embodiments of Structure 5 ⁇ include n is two, m is one, and R is hydrogen, n is three, m is zero, and R is hydrogen (in this embodiment, the terminal nitrogen (the D atom) may be the nitrogen of the ammo-modified ⁇ bose), and the use of R groups to increase solubility
- Preferred embodiments of Structure 6 include the first n is two, second n is one, m is zero, and all R groups are hydrogen, or the use of R groups to increase solubility
- Preferred embodiments of Structure 7 include the first n is three, the second n is from 1-3, with m being either 0 or 1 , and the use of R groups to increase solubility
- the conductive oligomer has the structure depicted in Structure 8
- C are carbon atoms
- n is an integer from 1 to 50
- m is 0 or 1
- J is a heteroatom selected from the group consisting of oxygen, nitrogen, silicon, phosphorus, sulfur, carbonyl or sulfoxide
- the G bond of each subunit may be the same or different than the G bonds of other subunits that is, alternating oligomers of alkene and acetylene bonds could be used, etc However, when G is an alkane bond, the number of alkane bonds in
- the m of Structure 8 is zero In a particularly preferred embodiment, m is zero and G is an alkene bond, as is depicted in Structure 9
- alkene oligomer of structure 9, and others depicted herein, are generally depicted in the preferred trans configuration, although oligomers of cis or mixtures of trans and cis may also be used As above, R groups may be added to alter the packing of the compositions on an electrode, the hydrophi city or hydrophobicity of the oligomer, and the flexibility, i e the rotational, torsional or longitudinal flexibility of the oligomer n is as defined above
- R is hydrogen, although R may be also alkyl groups and polyethylene glycols or derivatives
- the conductive oligomer may be a mixture of different types of oligomers, for example of structures 1 and 8
- the conductive oligomers may or may not have terminal groups Thus, in a preferred embodiment, there is no additional terminal group, and the conductive oligomer terminates with one of the groups depicted in Structures 1 to 9, for example, a B-D bond such as an acetylene bond Alternatively, in a preferred embodiment, a terminal group is added, sometimes depicted herein as "Q" A terminal group may be used for several reasons, for example, to contribute to the electronic availability of the conductive oligomer for detection of ETMs, or to alter the surface of the SAM for other reasons, for example to prevent non-specific binding
- the target analyte is a nucleic acid
- Preferred terminal groups include -NH 2 , -OH, -COOH, and alkyl groups such as -CH 3
- terminal groups may facilitate detection, and some may prevent non-specific binding
- the monolayer may further comprise insulator moieties
- insulator herein is meant a substantially nonconducting oligomer, preferably linear
- substantially nonconducting ' herein is meant that the insulator will not transfer electrons at 100 Hz The rate of electron transfer through the insulator is preferrably slower than the rate through the conductive oligomers described herein
- the insulators have a conductivity, S, of about 10 7 ⁇ 'c 1 or lower, with less than about 10 8 ⁇ 'cm 1 being preferred See generally Gardner et al , supra.
- insulators are alkyl or heteroalkyl oligomers or moieties with sigma bonds, although any particular insulator molecule may contain aromatic groups or one or more conjugated bonds
- heteroalkyl herein is meant an alkyl group that has at least one heteroatom, i e nitrogen, oxygen, sulfur, phosphorus, silicon or boron included in the chain
- the insulator may be quite similar to a conductive oligomer with the addition of one or more heteroatoms or bonds that serve to inhibit or slow, preferably substantially, electron transfer
- Suitable insulators include, but are not limited to, -(CH 2 ) n -, -(CRH) n -, and -
- the insulators may be substituted with R groups as defined herein to alter the packing of the moieties or conductive oligomers on an electrode, the hydrophi city or hydrophobicity of the insulator, and the flexibility, i e the rotational, torsional or longitudinal flexibility of the insulator
- R groups as defined herein to alter the packing of the moieties or conductive oligomers on an electrode, the hydrophi city or hydrophobicity of the insulator, and the flexibility, i e the rotational, torsional or longitudinal flexibility of the insulator
- branched alkyl groups may be used
- the insulators may contain terminal groups as outlined above, particularly to influence the surface of the monolayer
- the length of the species making up the monolayer will vary as needed As outlined above, it appears that binding of target analytes (for example, hybridization of nucleic acids) is more efficient at a distance from the surface
- the species to which capture binding ligands are attached (as outlined below, these can be either
- the monolayer may comprise different surface species, in a variety of configurations, although preferably the different species are chosen such that a reasonably uniform SAM can be formed
- the different species are chosen such that a reasonably uniform SAM can be formed
- preferred embodiments utilize surface species such as EFS including conductive oligomers that terminate below the surface of the rest of the monolayer, i.e. below the insulator layer, if used, or below some fraction of the other conductive oligomers
- the use of different species such as conductive oligomers may be done to facilitate monolayer formation, or to make monolayers with altered properties
- the actual combinations and ratios of the different species making up the monolayer can vary widely, and will depend on whether mechan ⁇ sm-1 or -2 is used, and whether rough or smooth surfaces are used Generally, three component systems are preferred for mechan ⁇ sm-2 systems, with the first species comprising a capture binding ligand containing species (termed a capture probe when the target analyte is a nucleic acid), attached to the electrode via an attachment linker (e.g. an insulator or conductive oligomer)
- the second species are EFS
- the third species are insulators
- the first species can comprise from about 90% to about
- first second third species can comprise from about 1 % to about 90%, with from about 20% to about 40% being preferred, and from about 15% to about 30% being especially preferred.
- preferred ratios of first second third species in SAM formation solvents are 2 2 1 for short targets, 1 3 1 for longer targets, with total thiol concentration (when used to attach these species, as is more fully outlined below) in the 500 ⁇ M to 1 mM range, and 833 ⁇ M being preferred
- two component systems can be used In one embodiment, for use in either mechan ⁇ sm-1 or mechan ⁇ sm-2 systems, the two components are the first and second species In this embodiment, the first species are the first and second species In this embodiment, the first species
- the deposition of the SAM is done using aqueous solvents
- aqueous solvents As is generally described in Steel et al , Anal Chem 70 4670 (1998), Herne et al , J Am Chem Soc 119 8916 (1997), and Fmklea, Electrochemistry of Organized Monolayers of Thiols and Related Molecules on Electrodes, from A J Bard, Electroanalvtical Chemistry A Series of Advances, Vol 20, Dekker N Y
- the covalent attachment of the monolayer-forming species and the capture binding ligand species to the electrode may be accomplished in a variety of ways, depending on the electrode and the composition of the moieties
- the attachment linkers with covalently attached nucleosides or nucleic acids as depicted herein are covalently attached to an electrode.
- one end or terminus of the attachment linker is attached to the nucleoside or nucleic acid, and the other is attached to an electrode
- the attachment linker may be desirable to have the attachment linker attached at a position other than a terminus, or even to have a branched attachment linker that is attached to an electrode at one terminus and to two or more nucleosides at other termini, although this is not preferred
- the attachment linker may be attached at two sites to the electrode, as is generally depicted in Structures 11-13
- some type of linker is used, as depicted below as "A" in Structure 10, where "X” is the EFS (e g conductive oligomer), "I” is an insulator and the hatched surface is the electrode Structure 10
- A is a linker or atom
- A may be a sulfur moiety when a gold electrode is used
- A may be a silicon (silane) moiety attached to the oxygen of the oxide (see for example Chen et al , Langmuir 10 3332-3337 (1994), Lenhard et al , J Electroanal Chem 78 195-201 (1977), both of which are expressly incorporated by reference)
- A may be an ammo moiety (preferably a primary amine, see for example Demhammer et al , Langmuir 10 1306-1313 (1994))
- preferred A moieties include, but are not limited to, silane moieties, sulfur moieties (including alkyl sulfur moieties), and ammo moieties
- epoxide type linkages with redox polymers such
- these moieties may be attached to the electrode with more than one A" moiety, the "A" moieties may be the same or different
- the electrode is a gold electrode
- "A" is a sulfur atom or moiety
- multiple sulfur atoms may be used to attach the species to the electrode, such as is generally depicted below in Structures 11 , 12 and 13 for conductive oligomers
- other such structures can be made In Structures 11 , 12 and 13, the A moiety is just a sulfur atom, but substituted sulfur moieties may also be used
- the species herein may also comprise a "Q" terminal group
- the electrode is a gold electrode, and attachment is via a sulfur linkage as is well known in the art, i e the A moiety is a sulfur atom or moiety Although the exact characteristics of the gold-sulfur attachment are not known, this linkage is considered covalent for the purposes of this invention
- a representative structure is depicted in Structure 14, using the Structure 3 conductive oligomer, although as for all the structures depicted herein, any surface species may be used Similarly, any of the surface species may also comprise terminal groups as described herein Structure 14 depicts the "A" linker as comprising just a sulfur atom, although additional atoms may be present (i e linkers from the sulfur to the conductive oligomer or substitution groups) In addition, Structure 14 shows the sulfur atom attached
- the electrode is a carbon electrode, i e a glassy carbon electrode, and attachment is via a nitrogen of an amine group
- a representative structure is depicted in Structure 15 Again, additional atoms may be present, i e Z type linkers and/or terminal groups
- the oxygen atom is from the oxide of the metal oxide electrode
- the Si atom may also contain other atoms, i e be a silicon moiety containing substitution groups Other attachments for
- SAMs to other electrodes are known in the art, see for example Napier et al , Langmuir, 1997, for attachment to indium tin oxide electrodes, and also the chemisorption of phosphates to an indium tin oxide electrode (talk by H Holden Thorpe, CHI conference, May 4-5, 1998)
- the SAMs of the invention can be made in a variety of ways, including deposition out of organic solutions and deposition out of aqueous solutions
- the methods outlined herein use a gold electrode as the example, although as will be appreciated by those in the art, other metals and methods may be used as well
- ITO indium-tm-oxide
- a gold surface is first cleaned
- cleaning procedures including, but not limited to, chemical cleaning or etchants (including Piranha solution (hydrogen peroxide/sulfu ⁇ c acid) or aqua regia (hydrochloric acid/nitric acid) electrochemical methods, flame treatment, plasma treatment or combinations thereof
- the SAM species are phosphonate-contaming species
- a deposition solution comprising a mixture of various SAM species in solution, generally thiol-containmg species
- Mixed monolayers that contain target analytes, particularly DNA are usually prepared using a two step procedure
- the thiolated DNA is deposited during the first deposition step (generally in the presence of at least one other monolayer-forming species) and the mixed monolayer formation is completed during the second step in which a second thiol solution minus DNA is added
- the second step frequently involves mild heating to promote monolayer reorganization
- the deposition solution is an organic deposition solution
- a clean gold surface is placed into a clean vial
- a binding ligand deposition solution in organic solvent is prepared in which the total thiol concentration is between micromolar to saturation, preferred ranges include from about 1 ⁇ M to 10 mM, with from about 400 uM to about 1 0 mM being especially preferred
- the deposition solution contains thiol modified DNA (i e nucleic acid attached to an attachment linker) and thiol diluent molecules (either conductive oligomers or insulators, with the latter being preferred)
- the ratio of DNA to diluent (if present) is usually between 1000 1 to 1 1000, with from about 10 1 to about 1 10 being preferred and 1 1 being especially preferred
- the preferred solvents are tetrahydrofuran (THF), acetonitrile, dimethylforamide (DMF), ethanol, or mixtures thereof, generally any solvent of sufficient polarity to dissolve the capture ligand
- the gold substrate is allowed to incubate at ambient temperature or slightly above ambient temperature for a period of time ranging from seconds to hours, with 5-30 minutes being preferred
- the deposition solution is removed and a solution of diluent molecule only (from about 1 ⁇ M to 10 mM, with from about 100 uM to about 1 0 mM being preferred) in organic solvent is added
- the gold substrate is allowed to incubate at room temperature or above room temperature for a period of time (seconds to days, with from about 10 minutes to about 24 hours being preferred)
- the gold sample is removed from the solution, rinsed in clean solvent and used
- an aqueous deposition solution is used as above, a clean gold surface is placed into a clean vial A DNA deposition solution in water is prepared in which the total thiol concentration is between about 1 uM and 10 mM, with from about 1 ⁇ M to about 200 uM being preferred The aqueous solution frequently has salt present (up to saturation, with approximately 1 M being preferred), however pure water can be used
- the deposition solution contains thiol modified DNA and often a thiol diluent molecule The ratio of DNA to diluent is usually between 1000 1 to 1 1000, with from about 10 1 to about 1 10 being preferred and 1 1 being especially preferred
- the DNA deposition solution is added to the vial in such a volume so as to completely cover the electrode surface
- the gold substrate is allowed to incubate at ambient temperature or slightly above ambient temperature for 1-30 minutes with 5 minutes usually being sufficient After the initial incubation, the deposition solution is removed and a solution of diluent molecule only (10 uM -1 0
- a circuit board is used as the substrate for the gold electrodes
- Formation of the SAMs on the gold surface is generally done by first cleaning the boards, for example in a 10% sulfuric acid solution for 30 seconds, detergent solutions, aqua regia, plasma, etc , as outlined herein Following the sulfuric acid treatment, the boards are washed, for example via immersion in two Milli-Q water baths for 1 minute each The boards are then dried, for example under a stream of nitrogen Spotting of the deposition solution onto the boards is done using any number of known spotting systems, generally by placing the boards on an X-Y table, preferably in a humidity chamber
- the size of the spotting drop will vary with the size of the electrodes on the boards and the equipment used for delivery of the solution, for example, for 250 ⁇ M size electrodes, a 30 nanohter drop is used The volume should be sufficient to cover the electrode surface completely The drop is incubated at room temperature for a period of time (sec to overnight, with 5 minutes preferred) and then the drop
- the detection electrode further comprises a capture binding ligand, preferably covalently attached
- binding ligand or ' binding species herein is meant a compound that is used to probe for the presence of the target analyte, that will bind to the target analyte
- binding ligand or ' binding species herein is meant a compound that is used to probe for the presence of the target analyte, that will bind to the target analyte
- there are at least two binding ligands used per target analyte molecule a “capture” or “anchor” binding ligand (also referred to herein as a “capture probe' , particularly in reference to a nucleic acid binding ligand) that is attached to the detection electrode as described herein, and a soluble binding ligand (also referred to herein as a "label probe” or ' signalling probe", when nucleic acids are used), that binds independently to the target analyte, and either directly or indirectly comprises at
- the capture binding ligand allows the attachment of a target analyte to the biosensor electrode, for the purposes of detection
- attachment of the target analyte to the capture binding ligand may be direct (i e the target analyte binds to the capture binding ligand) or indirect (one or more capture extender ligands may be used)
- the binding is specific, and the binding ligand is part of a binding pair
- specifically bind herein is meant that the ligand binds the analyte, with specificity sufficient to differentiate between the analyte and other components or contaminants of the test sample
- the systems may use different binding ligands, for example an array of different ligands, and detection of any particular analyte is via its "signature" of binding to a panel of binding ligands, similar to the manner in which "electronic noses" work
- the binding should be sufficient to allow the analyte to remain bound under the conditions of the assay, including wash steps to remove non-specific binding
- the binding constants of the analyte to the binding ligand will be at least about 10 ⁇ to 10 6
- the composition of the binding ligand will depend on the composition of the target analyte Binding ligands to a wide variety of analytes are known or can be readily found using known techniques
- the binding ligand is generally a substantially complementary nucleic acid
- nucleic acid "aptomers" can be developed for binding to virtually any target analyte
- the analyte may be a nucleic acid binding protein and the capture binding ligand is either a single-stranded or double-stranded nucleic acid
- the binding ligand may be a nucleic acid binding protein when the analyte is
- the binding ligand is a nucleic acid
- preferred compositions and techniques are outlined in U S Patent Nos 5,591 ,578, 5,824,473, 5,770,369, 5,705,348, 5,780,234, and 5,952,172 WO98/20162, W099/37819, PCT US98/12430, PCT US99/01703, PCT US98/12082, PCT/US99/14191 , PCT/US99/21683, PCT/US99/25464, PCT US99/10104, and U S S N.s 09/135,183, 09/295,691 and 09/306,653, all of which are hereby expressly incorporated by reference
- the method of attachment of the capture binding ligands to the attachment linker will generally be done as is known in the art, and will depend on both the composition of the attachment linker and the capture binding ligand
- the capture binding ligands are attached to the attachment linker through the use of functional groups on each that can then be used for attachment
- Preferred functional groups for attachment are am o groups, carboxy groups, oxo groups and thiol groups
- Z Linkers
- Z linkers are well known in the art, for example, homo-or hetero-b functional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference)
- Preferred Z linkers include, but are not limited to, alkyl groups (including substituted alkyl groups and alkyl groups containing heteroatom moieties), with short alkyl groups, esters,
- capture binding ligands comprising proteins, lectins, nucleic acids, small organic molecules, carbohydrates, etc can be added
- a preferred embodiment utilizes protemaceous capture binding ligands
- any number of techniques may be used to attach a protemaceous capture binding ligand to an attachment linker
- any number of techniques may be used to attach a protemaceous capture binding ligand to an attachment linker
- any wide variety of techniques are known to add moieties to proteins
- a preferred embodiment utilizes nucleic acids as the capture binding ligand While most of the following discussion focuses on nucleic acids, as will be appreciated by those in the art, many of the techniques outlined below apply in a similar manner to non-nucleic acid systems as well
- the capture probe nucleic acid is covalently attached to the electrode, via an "attachment linker", that can be any surface species, including either a conductive oligomer (required for mechan ⁇ sm-1 systems) or an insulator
- an “attachment linker” can be any surface species, including either a conductive oligomer (required for mechan ⁇ sm-1 systems) or an insulator
- covalently attached herein is meant that two moieties are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds
- nucleic acid or other binding ligand
- the other end is attached to the electrode
- any of structures depicted herein may further comprise a nucleic acid effectively as a terminal group
- the present invention provides compositions comprising nucleic acids covalently attached to electrodes as is generally depicted below in Structure
- F is a linkage that allows the covalent attachment of the electrode and the conductive oligomer or insulator, including bonds, atoms or linkers such as is described herein, for example as "A”, defined below
- F 2 is a linkage that allows the covalent attachment of the surface species to the nucleic acid, and may be a bond, an atom or a linkage as is herein described
- F 2 may be part of the conductive oligomer, part of the insulator, part of the nucleic acid, or exogeneous to both, for example, as defined herein for "Z"
- the capture probe nucleic acid is covalently attached to the electrode via a conductive oligomer
- the covalent attachment of the nucleic acid and the conductive oligomer may be accomplished in several ways In a preferred embodiment, the attachment is via attachment to the base of the nucleoside, via attachment to the backbone of the nucleic acid (either the ⁇ bose, the phosphate, or to an analogous group of a nucleic acid analog backbone), or via a transition metal ligand, as described below
- the techniques outlined below are generally described for naturally occunng nucleic acids, although as will be appreciated by those in the art, similar techniques may be used with nucleic acid analogs, and in some cases with other binding ligands
- the attachment linkers are attached to the base of a nucleoside of the nucleic acid This may be done in several ways, depending on the attachment linker, as is described below In one embodiment, the attachment linker is attached to a terminal nucleoside, i e either the 3' or 5' nucleoside of the nucleic acid Alternatively, the attachment linker is attached to an internal nucleoside
- the point of attachment to the base will vary with the base Generally, attachment at any position is possible In some embodiments, for example when the probe containing the ETMs may be used for hybridization (i e mechan ⁇ sm-1 systems) , it is preferred to attach at positions not involved in hydrogen bonding to the complementary base Thus, for example, generally attachment is to the 5 or 6 position of py ⁇ midines such as undine, cytosme and thymine For pu ⁇ nes such as adenine and guanine, the linkage is preferably via the 8 position Attachment to non-standard bases is preferably done at the comparable positions
- the attachment is direct, that is, there are no intervening atoms between the attachment linker and the base
- attachment linkers that are conductive oligomers with terminal acetylene bonds are attached directly to the base Structure 18 is an example of this linkage, using a Structure 3 conductive oligomer and undine as the base, although other bases and conductive oligomers or attachment linkers can be used as will be appreciated by those in the art
- pentose structures depicted herein may have hydrogen, hydroxy, phosphates or other groups such as ammo groups attached
- pentose and nucleoside structures depicted herein are depicted non-conventionally, as mirror images of the normal rendering
- the pentose and nucleoside structures may also contain additional groups, such as protecting groups, at any position, for example as needed during synthesis
- the base may contain additional modifications as needed, i e the carbonyl or amine groups may be altered or protected
- the attachment is any number of different Z linkers, including amide and amine linkages, as is generally depicted in Structure 19 using undine as the base and a Structure 3 oligomer
- Z is a linker
- Z is a short linker of about 1 to about 10 atoms, with from 1 to 5 atoms being preferred, that may or may not contain alkene, alkynyl, amine, amide, azo, imine, etc , bonds
- Linkers are known in the art, for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages
- Preferred Z linkers include, but are not limited to, alkyl groups (including substituted alkyl groups and alkyl groups containing heteroatom moieties), with short alkyl groups, esters, amide, amine, epoxy groups and ethylene glycol and derivatives being preferred, with propyl, acetylene, and C 2 alkene being especially preferred Z may also be a sulfone group, forming sulfonamide linkages as discussed below
- the attachment of the nucleic acid and the attachment linker is done via attachment to the backbone of the nucleic acid This may be done in a number of ways, including attachment to a ⁇ bose of the ⁇ bose-phosphate backbone, or to the phosphate of the backbone, or other groups of analogous backbones
- the site of attachment in this embodiment may be to a 3' or 5' terminal nucleotide, or to an internal nucleotide, as is more fully described below
- the attachment linker is attached to the ⁇ bose of the ⁇ bose-phosphate backbone This may be done in several ways As is known in the art, nucleosides that are modified at either the 2' or 3' position of the ⁇ bose with ammo groups, sulfur groups, silicone groups, phosphorus groups, or oxo groups can be made (Imazawa et al , J Org Chem , 44 2039 (1979), Hobbs et al , J
- a preferred embodiment utilizes amino-modified nucleosides These amino-modified ⁇ boses can then be used to form either amide or amine linkages to the attachment linkers
- the am o group is attached directly to the ⁇ bose, although as will be appreciated by those in the art, short linkers such as those described herein for "Z" may be present between the ammo group and the ⁇ bose
- an amide linkage is used for attachment to the ⁇ bose
- m is zero and thus the conductive oligomer terminates in the amide bond
- the nitrogen of the ammo group of the amino-modified ⁇ bose is the "D" atom of the conductive oligomer
- Structure 20 has the terminal bond fixed as an amide bond
- a heteroatom linkage is used, i e oxo, amine, sulfur, etc
- a preferred embodiment utilizes an amine linkage
- the nitrogen of the amino-modified ⁇ bose may be the "D" atom of the conductive oligomer when the Structure 3 conductive oligomer is used
- Structures 21 and 22 depict nucleosides with the Structures 3 and 9 conductive oligomers, respectively, using the nitrogen as the heteroatom, athough other heteroatoms can be used
- both m and t are not zero
- a preferred Z here is a methylene group, or other aliphatic alkyl linkers One, two or three carbons in this position are particularly useful for synthetic reasons
- Suitable linkers include methylene and ethylene
- the attachment linker is covalently attached to the nucleic acid via the phosphate of the ⁇ bose-phosphate backbone (or analog) of a nucleic acid
- the attachment is direct, utilizes a linker or via an amide bond
- Structure 23 depicts a direct linkage
- Structure 24 depicts linkage via an amide bond (both utilize the Structure 3 conductive oligomer, although Structure 8 conductive oligomers and other attachment linkers are also possible)
- Structures 23 and 24 depict the conductive oligomer in the 3' position, although the 5' position is also possible
- both Structures 23 and 24 depict naturally occurring phosphodiester bonds, although as those in the art will appreciate non-standard analogs of phosphodiester bonds may also be used Structure 23
- Structure 24 depicts a preferred embodiment, wherein the terminal B-D bond is an amide bond, the terminal Y is not present, and Z is a linker, as defined herein Structure 24
- the attachment linker is covalently attached to the nucleic acid via a transition metal ligand
- the attachment linker is covalently attached to a ligand which provides one or more of the coordination atoms for a transition metal
- the ligand to which the attachment linker is attached also has the nucleic acid attached, as is generally depicted below in Structure 25
- the attachment linker is attached to one ligand, and the nucleic acid is attached to another ligand, as is generally depicted below in Structure 26 using conductive oligomers as the attachment linker
- the attachment linker is covalently attached to the nucleic acid
- Both of these structures depict Structure 3 conductive oligomers, although other oligomers may be utilized Structures 25 and 26 depict two representative structures
- M is a metal atom, with transition metals being preferred Suitable transition metals for use in the invention include, but are not limited to, cadmium (Cd), copper (Cu), cobalt (Co), palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru), rhodium (Rh), osmium (Os), rhenium (Re), platinium (Pt), scandium (Sc), titanium (Ti), Vanadium (V), chromium (Cr), manganese (Mir), nickel (Ni), Molybdenum (Mo), technetium (Tc), tungsten (W), and indium (Ir) That is, the first series of transition metals, the platinum metals (Ru, Rh, Pd, Os, Ir and Pt), along with Fe, Re, W, Mo and Tc, are preferred Particularly preferred are ruthenium, rhenium, osmium, platinium, cobal
- L are the co-hgands, that provide the coordination atoms for the binding of the metal ion
- the number and nature of the co-hgands will depend on the coordination number of the metal ion
- Mono-, di- or polydentate co-hgands may be used at any position
- r may range from zero (when all coordination atoms are provided by the other two ligands) to four, when all the co-hgands are monodentate
- r will be from 0 to 8, depending on the coordination number of the metal ion and the choice of the other ligands
- the metal ion has a coordination number of six and both the ligand attached to the conductive oligomer and the ligand attached to the nucleic acid are at least bidentate, that is, r is preferably zero, one (i e the remaining co-ligand is bidentate) or two (two monodentate co-hgands are used)
- Suitable ligands can be the same or different Suitable ligands fall into two categories ligands which use nitrogen, oxygen, sulfur, carbon or phosphorus atoms (depending on the metal ion) as the coordination atoms (generally referred to in the literature as sigma ( ⁇ ) donors) and organometalhc ligands such as metallocene ligands (generally referred to in the literature as pi ( ⁇ ) donors, and depicted herein as L Suitable nitrogen donating ligands are well known in the art and include, but are not limited to, NH 2 , NHR, NRR pyridine, pyrazme, isonicotinamide, imidazole, bipyndine and substituted de ⁇ vatives of bipy ⁇ dine, terpy ⁇ dine and substituted derivatives, phenanthrohnes, particularly 1 ,10-phenanthrohne (abbreviated phen) and substituted derivatives of phenanthrohne
- Suitable sigma donating ligands using carbon, oxygen, sulfur and phosphorus are known in the art
- suitable sigma carbon donors are found in Cotton and Wilkenson, Advanced Organic
- oxygen ligands include crown ethers, water and others known in the art Phosphmes and substituted phosphmes are also suitable, see page 38 of Cotton and Wilkenson
- oxygen, sulfur, phosphorus and nitrogen-donating ligands are attached in such a manner as to allow the heteroatoms to serve as coordination atoms
- organometalhc ligands are used
- organometalhc compounds are used
- organometalhc ligands include cyclic aromatic compounds such as the cyclopentadienide ion [C 5 H 5 (-1 )] and various ring substituted and ring fused derivatives, such as the indenyhde (-1 ) ion, that yield a class of b ⁇ s(cyclopentad ⁇ eyl) metal compounds, (i e the metallocenes), see for example Robins et al , J Am Chem Soc 104 1882-1893 (1982), and Gassman et al , J Am Chem Soc 108 4228-4229 (1986), incorporated by reference Of these, ferrocene [(C 5 H 5 H 5
- Organometalhc ligands include cyclic arenes such as benzene, to yield b ⁇ s(arene)metal compounds and their ring substituted and ring fused derivatives, of which b ⁇ s(benzene)chrom ⁇ um is a prototypical example,
- Other acyclic ⁇ - bonded ligands such as the allyl(-1 ) ion, or butadiene yield potentially suitable organometalhc compounds, and all such ligands, in conjuction with other ⁇ -bonded and ⁇ -bonded ligands constitute the general class of organometalhc compounds in which there is a
- the ligand is generally attached via one of the carbon atoms of the organometalhc ligand, although attachment may be via other atoms for heterocyclic ligands
- Preferred organometalhc ligands include metallocene ligands, including substituted derivatives and the metalloceneophanes (see page 1174 of Cotton and Wilkenson, supra)
- metallocene ligands such as methylcyclopentadienyl, with multiple methyl groups being preferred, such as pentamethylcyclopentadienyl, can be used to increase the stability of the metallocene
- methylcyclopentadienyl with multiple methyl groups being preferred, such as pentamethylcyclopentadienyl
- pentamethylcyclopentadienyl can be used to increase the stability of the metallocene
- only one of the two metallocene ligands of a metallocene are denvatized
- any combination of ligands may be used Preferred combinations include: a) all ligands are nitrogen donating ligands, b) all ligands are organometalhc ligands; and c) the ligand at the terminus of the conductive oligomer is a metallocene ligand and the ligand provided by the nucleic acid is a nitrogen donating ligand, with the other ligands, if needed, are either nitrogen donating ligands or metallocene ligands, or a mixture
- These combinations are depicted in representative structures using the conductive oligomer of Structure 3 are depicted in Structures 27 (using phenanthrohne and am o as representative ligands), 28 (using ferrocene as the metal-hgand combination) and 29 (using cyclopentadienyl and ammo as representative ligands)
- the ligands used in the invention show altered fluoroscent properties depending on the redox state of the chelated metal ion As described below, this thus serves as an additional mode of detection of electron transfer between the ETM and the electrode
- the ligand attached to the nucleic acid is an am o group attached to the 2' or 3' position of a ⁇ bose of the ⁇ bose-phosphate backbone
- This ligand may contain a multiplicity of am o groups so as to form a polydentate ligand which binds the metal ion
- Other preferred ligands include cyclopentadiene and phenanthro ne
- metal ions to connect the nucleic acids can serve as an internal control or calibration of the system, to evaluate the number of available nucleic acids on the surface
- metal ions are used to connect the nucleic acids to the attachment linker, it is generally desirable to have this metal ion complex have a different redox potential than that of the ETMs used in the rest of the system, as described below This is generally true so as to be able to distinguish the presence of the capture probe from the presence of the target sequence This may be useful for identification, calibration and/or quantification
- the amount of capture probe on an electrode may be compared to the amount of hybridized double stranded nucleic acid to quantify the amount of target sequence in a sample This is quite significant to serve as an internal control of the sensor or system This allows a measurement either prior to the addition of target or after, on the same molecules that will be used for detection, rather than rely on a similar but different control system
- the actual molecules that will be used for the detection can be quantified
- there may be one or more different capture probe species on the surface there may be one type of capture probe, or one type of capture probe extender, as is more fully described below
- different capture probes, or one capture probes with a multiplicity of different capture extender probes can be used
- tissue collection devices comprising biosensors with substrates comprising at least one electrode comprising monolayers and capture binding ligands, useful in target analyte detection systems of collected samples
- the compositions further comprise a solution or soluble binding ligand, although as is more fully described below, for mechan ⁇ sm-1 systems, the ETMs may be added in the form of non-covalently attached hybridization indicators
- Solution binding ligands are similar to capture binding ligands, in that they bind, preferably specifically, to target analytes
- the solution binding ligand may be the same or different from the capture binding ligand
- the solution binding ligands are not directed attached to the surface, although in some embodiments they may be
- the solution binding ligand either directly comprises a recruitment linker that comprises at least one ETM or the recruitment linker binds, either directly or indirectly to the solution binding ligand
- solution binding ligands or “soluble binding ligands” or “signalling ligands” or “signal carriers” or
- label probes or “label binding ligands” with recruitment linkers comprising covalently attached ETMs are provided That is, one portion of the label probe or solution binding ligand directly or indirectly binds to the target analyte, and one portion comprises a recruitment linker comprising covalently attached ETMs In some systems, for example in mechan ⁇ sm-1 nucleic acid systems, these may be the same Similarly, for mechan ⁇ sm-1 systems, the recruitment linker comprises nucleic acid that will hybridize to detection probes
- ETMs electron donor moiety
- ETMs electron acceptor moiety
- ETMs electron acceptor moiety
- ETMs electron acceptor moiety
- grammatical equivalents herein refers to molecules capable of electron transfer under certain conditions It is to be understood that electron donor and acceptor capabilities are relative, that is, a molecule which can lose an electron under certain experimental conditions will be able to accept an electron under different experimental conditions It is to be understood that the number of possible electron donor moieties and electron acceptor moieties is very large, and that one skilled in the art of electron transfer compounds will be able to utilize a number of compounds in the present invention
- Preferred ETMs include, but are not limited to, transition metal complexes, organic ETMs, and electrodes
- the ETMs are transition metal complexes
- Transition metals are those whose atoms have a partial or complete d shell of electrons Suitable transition metals for use in the invention are listed above
- transition metals are complexed with a variety of ligands, L, defined above, to form suitable transition metal complexes, as is well known in the art
- organic electron donors and acceptors may be covalently attached to the nucleic acid for use in the invention
- organic molecules include, but are not limited to, ⁇ boflavin, xanthene dyes, azme dyes, ac ⁇ dine orange, ⁇ /,.V-d ⁇ metr.yl-2,7- diazapyrenium dichlo ⁇ de (DAP 2+ ), methylviologen, ethidium bromide, quinones such as N,N'- d ⁇ methylanthra(2,1 ,9-det6,5,10-d'e'f)d ⁇ soqu ⁇ nohne dichlo ⁇ de (ADIQ 2* ), porphy ⁇ ns ([meso-tetrak ⁇ s(N- methyl-x-py ⁇ d ⁇ n ⁇ um)porphy ⁇ n tetrachlo ⁇ de], varlamme blue B hydrochloride, Bindschedler's green, 2,6- dichloro
- the electron donors and acceptors are redox proteins as are known in the art
- ETMs are metallocenes, with ferrocene, including derivatives, being particularly preferred
- a plurality of ETMs are used The use of multiple ETMs provides signal amplification and thus allows more sensitive detection limits While the use of multiple ETMs on nucleic acids that hybridize to complementary strands can cause decreases in T m s of the hybridization complexes depending on the number, site of attachment and spacing between the multiple ETMs, this is not a factor when the ETMs are on the recruitment linker (i e "mechan ⁇ sm-2" systems), since this does not hybridize to a complementary sequence Accordingly, pluralities of ETMs are preferred, with at least about 2 ETMs per recruitment linker being preferred, and at least about 10 being particularly preferred and at least about 20 to 50 being especially preferred In some instances, very large numbers of ETMs (50 to 1000) can be used
- solution binding ligands, or label probes, with covalently attached ETMs are provided
- the method of attachment of the ETM to the solution binding ligand will vary depending on the mode of detection (i e mechan ⁇ sm-1 or -2 systems) and the composition of the solution binding ligand
- the portion of the solution binding ligand (or label probe) that comprises the ETM is referred to as a ' recruitment linker and can comprise either nucleic acid or non-nucleic acid
- the recruitment linker must be nucleic acid
- the recruitment linker is nucleic acid (including analogs), and attachment of the ETMs can be via (1 ) a base, (2) the backbone, including the ⁇ bose, the phosphate, or comparable structures in nucleic acid analogs, (3) nucleoside replacement, described below, or (4) metallocene polymers, as described below
- the recruitment linker is non-nucleic acid, and can be either a metallocene polymer or an alkyl-type polymer (including heteroalkyl, as is more fully described below) containing ETM substitution groups
- the recruitment linker is a nucleic acid, and comprises covalently attached ETMs
- the ETMs may be attached to nucleosides within the nucleic acid in a variety of positions
- Preferred embodiments include, but are not limited to, (1 ) attachment to the base of the nucleoside, (2) attachment of the ETM as a base replacement, (3) attachment to the backbone of the nucleic acid, including either to a ⁇ bose of the nbose-phosphate backbone or to a phosphate moiety, or to analogous structures in nucleic acid analogs, and (4) attachment via metallocene polymers
- the recruitment linker is nucleic acid
- secondary label probes that have a first portion that will hybridize to a portion of the primary label probes and a second portion comprising a recruitment linker as is defined herein This is similar to the use of an amplifier probe, except that both the primary and the secondary label probes comprise ETMs
- the ETM is attached to the base of a nucleoside as is generally outlined above for attachment of the conductive oligomer Attachment can be to an internal nucleoside or a terminal nucleoside
- the covalent attachment to the base will depend in part on the ETM chosen, but in general is similar to the attachment of conductive oligomers to bases, as outlined above Attachment may generally be done to any position of the base
- the ETM is a transition metal complex, and thus attachment of a suitable metal ligand to the base leads to the covalent attachment of the ETM Alternatively, similar types of linkages may be used for the attachment of organic ETMs, as will be appreciated by those in the art
- the C4 attached ammo group of cytosme, the C6 attached ammo group of adenine, or the C2 attached am o group of guanine may be used as a transition metal ligand
- Ligands containing aromatic groups can be attached via acetylene linkages as is known in the art (see Comprehensive Organic Synthesis, Trost et al , Ed , Pergamon Press, Chapter 2 4 Coupling
- Structure 30 depicts a representative structure in the presence of the metal ion and any other necessary ligands; Structure 30 depicts undine, although as for all the structures herein, any other base may also be used. Structure 30
- L a is a ligand, which may include nitrogen, oxygen, sulfur or phosphorus donating ligands or organometalhc ligands such as metallocene ligands.
- Suitable L a ligands include, but not limited to, phenanthrohne, imidazole, bpy and terpy L r and M are as defined above.
- a linker (“Z") may be included between the nucleoside and the ETM.
- the linkage may be done using a linker, which may utilize an amide linkage (see generally Telser et al , J. Am Chem. Soc. 111 :7221-7226 (1989); Telser et al., J. Am. Chem. Soc. 1 11 :7226-7232 (1989), both of which are expressly incorporated by reference).
- a linker which may utilize an amide linkage (see generally Telser et al , J. Am Chem. Soc. 111 :7221-7226 (1989); Telser et al., J. Am. Chem. Soc. 1 11 :7226-7232 (1989), both of which are expressly incorporated by reference).
- L is a ligand as defined above, with L r and M as defined above as well
- L is ammo, phen, byp and terpy
- the ETM attached to a nucleoside is a metallocene, i e the L and L r of Structure 31 are both metallocene ligands, L m , as described above Structure 32 depicts a preferred embodiment wherein the metallocene is ferrocene, and the base is undine, although other bases may be used
- Preferred metallocenes include ferrocene, cobaltocene and osmiumocene
- the ETM is attached to a ⁇ bose at any position of the nbose-phosphate backbone of the nucleic acid, i.e either the 5' or 3' terminus or any internal nucleoside Ribose in this case can include ribose analogs
- nucleosides that are modified at either the 2' or 3' position of the ribose can be made, with nitrogen, oxygen, sulfur and phosphorus-containing modifications possible Amino-modified and oxygen-modified ribose is preferred See generally PCT publication WO 95/15971 , incorporated herein by reference
- modification groups may be used as a transition metal ligand, or as a chemically functional moiety for attachment of other transition metal ligands and organometalhc ligands, or organic electron donor moieties as will be appreciated by those in the art
- a linker such as depicted herein for "Z" may be used as well, or a
- a metallocene serves as the ETM, and is attached via an amide bond as depicted below in Structure 33
- the examples outline the synthesis of a preferred compound when the metallocene is ferrocene
- amine linkages are used, as is generally depicted in Structure 34
- Z is a linker, as defined herein, with 1-16 atoms being preferred, and 2-4 atoms being particularly preferred, and t is either one or zero
- oxo linkages are used, as is generally depicted in Structure 35
- an ETM is attached to a phosphate at any position of the nbose-phosphate backbone of the nucleic acid
- phosphodiester bond analogs such as phosphoramide or phosphoramidite linkages may be incorporated into a nucleic acid, where the heteroatom (i e nitrogen) serves as a transition metal ligand (see PCT publication WO 95/15971 , incorporated by reference)
- the conductive oligomers depicted in Structures 23 and 24 may be replaced by ETMs
- the composition has the structure shown in Structure 36
- Alkyl-based linkers can be used to create multiple branching structures comprising one or more ETMs at the terminus of each branch Generally, this is done by creating branch points containing multiple hydroxy groups, which optionally can then be used to add additional branch points The terminal hydroxy groups can then be used in phosphoramidite reactions to add ETMs, as is generally done below for the nucleoside replacement and metallocene polymer reactions
- an ETM such as a metallocene is used as a ' nucleoside replacement", serving as an ETM
- the distance between the two cyclopentadiene rings of ferrocene is similar to the orthongonal distance between two bases in a double stranded nucleic acid
- Other metallocenes in addition to ferrocene may be used, for example, air stable metallocenes such as those containing cobalt or ruthenium
- metallocene moieties may be incorporated into the backbone of a nucleic acid, as is generally depicted in Structure 37 (nucleic acid with a nbose-phosphate backbone) and Structure 38 (peptide nucleic acid backbone) Structures 37 and 38 depict ferrocene, although as will be appreciated by those in the art, other metallocenes may be used as well
- air stable metallocenes are preferred, including metallocenes utilizing ruthenium and cobalt as the metal
- Z is a linker as defined above, with generally short, alkyl groups, including heteroatoms such as oxygen being preferred Generally, what is important is the length of the linker, such that minimal perturbations of a double stranded nucleic acid is effected, as is more fully described below Thus, methylene, ethylene, ethylene glycols, propylene and butylene are all preferred, with ethylene and ethylene glycol being particularly preferred.
- each Z linker may be the same or different Structure 37 depicts a nbose-phosphate backbone, although as will be appreciated by those in the art, nucleic acid analogs may also be used, including ribose analogs and phosphate bond analogs
- ETMs in addition to metallocenes, as nucleoside replacements or in polymer embodiments, described below
- the ETM is a transition metal complex other than a metallocene, comprising one, two or three (or more) ligands
- the ligands can be functionahzed as depicted for the ferrocene to allow the addition of phosphoramidite groups
- Particularly preferred in this embodiment are complexes comprising at least two ring (for example, aryl and substituted aryl) ligands, where each of the ligands comprises functional groups for attachment via phosphoramidite chemistry
- this type of reaction creating polymers of ETMs either as a portion of the backbone of the nucleic acid or as "side groups" of the nucleic acids, to allow amplification of the signals generated herein,
- nucleic acid analogs are made that is, the invention provides nucleic acids having a backbone comprising at least one metallocene
- a metallocene such as ferrocene (or other ETM)
- nucleic acids having a backbone comprising at least one metallocene
- two nucleic acids each made up of a traditional nucleic acid or analog may be covalently attached to each other via a metallocene
- a metallocene derivative or substituted metallocene is provided, wherein each of the two aromatic rings of the metallocene has a nucleic acid substitutent group
- metallocene polymers that is, there are areas of metallocene polymers within the backbone
- nucleic acid substitutent groups it is also desirable in some instances to add additional substituent groups to one or both of the aromatic rings of the metallocene (or ETM)
- additional substituent groups to one or both of the aromatic rings of the metallocene (or ETM)
- these nucleoside replacements are generally part of probe sequences to be hybridized with a substantially complementary nucleic acid, for example a target sequence or another probe sequence
- substitutent groups to the metallocene rings to facilitate hydrogen bonding to the base or bases on the opposite strand
- substitutent groups include, but are not limited to, amide groups, amine groups, carboxylic acids, and alcohols, including substituted alcohols
- these substitutent groups can be attached via linkers as well, although in general this is not preferred
- substituent groups on an ETM may be added to alter the redox properties of the ETM
- substituent groups on an ETM may be added to alter the redox properties of the ETM
- substituent groups on the metallocene may allow two different ETMs to be distinguished
- the intermediate components are also provided.
- the invention provides phosphoramidite metallocenes, as generally depicted in Structure 39
- PG is a protecting group, generally suitable for use in nucleic acid synthesis, with DMT, MMT and TMT all being preferred
- the aromatic rings can either be the rings of the metallocene, or aromatic rings of ligands for transition metal complexes or other organic ETMs
- the aromatic rings may be the same or different, and may be substituted as discussed herein
- Structure 40 depicts the ferrocene derivative
- Structure 41 depicts the ferrocene peptide nucleic acid (PNA) monomer, that can be added to PNA synthesis as is known in the art
- the PG protecting group is suitable for use in peptide nucleic acid synthesis, with MMT, boc and Fmoc being preferred
- the ETMs are attached as polymers, for example as metallocene polymers, in a "branched" configuration similar to the "branched DNA” embodiments herein and as outlined in U S Patent No 5,124,246, using modified functionahzed nucleotides
- the general idea is as follows A modified phosphoramidite nucleotide is generated that can ultimately contain a free hydroxy group that can be used in the attachment of phosphoramidite ETMs such as metallocenes This free hydroxy group could be on the base or the backbone, such as the ribose or the phosphate (although as will be appreciated by those in the art, nucleic acid analogs containing other structures can also be used)
- the modified nucleotide is incorporated into a nucleic acid, and any hydroxy protecting groups are removed, thus leaving the free hydroxyl
- a phosphoramidite ETM such as a metallocene
- the 2' position of a ribose of a phosphoramidite nucleotide is first functionahzed to contain a protected hydroxy group, in this case via an oxo-linkage, although any number of linkers can be used, as is generally described herein for Z linkers
- the protected modified nucleotide is then incorporated via standard phosphoramidite chemistry into a growing nucleic acid
- the protecting group is removed, and the free hydroxy group is used, again using standard phosphoramidite chemistry to add a phosphoramidite metallocene such as ferrocene
- a similar reaction is possible for nucleic acid analogs
- nucleic acid analogs For example, using peptide nucleic acids and the metallocene monomer shown in Structure 41 , peptide nucleic acid structures containing metallocene polymers could be generated
- the present invention provides recruitment linkers of nucleic acids comprising ' branches" of metallocene polymers
- Preferred embodiments also utilize metallocene polymers from one to about 50 metallocenes in length, with from about 5 to about 20 being preferred and from about 5 to about 10 being especially preferred
- any combination of ETM attachments may be done
- clusters of nucleosides containing ETMs can decrease the Tm of hybridization of the probe to its target sequence, thus in general, for mechan ⁇ sm-1 systems, the ETMs are spaced out over the length of the sequence, or only small numbers of them are used
- the ⁇ ETM is a hybridization indicator
- Hybridization indicators serve as an ETM that will preferentially associate with double stranded nucleic acid is added, usually reversibly, similar to the method of Millan et al , Anal Chem 65 2317-2323 (1993) Millan et al , Anal Chem 662943-2948 (1994), both of which are hereby expressly incorporated by reference
- increases in the local concentration of ETMs, due to the association of the ETM hybridization indicator with double stranded nucleic acid at the surface can be monitored using the monolayers comprising the conductive oligomers
- Hybridization indicators include intercalators and minor and/or major groove binding moieties
- intercalators may be used, since intercalation generally only occurs in the presence of double stranded nucleic acid, only in the presence of double stranded nucleic acid will the ETMs concentrate Intercalating transition metal complex ETMs are known in the art Similarly, major or minor groove binding moieties, such as methylene blue, may also be used in this embodiment
- the biosensors of the invention may be used in virtually any method that relies on electrochemical detection of target analytes, with particular utility in nucleic acid detection
- the methods and compositions of the invention can be used in nucleic acid detection methods that rely on the detection of ETMs that are inherent to the target analyte
- the guanine bases of nucleic acid can be detected via changes in the redox state, i e guanine oxidation by ruthenium complexes
- the methods of the invention find use in detection systems that utilize copper surfaces as catalytic electrodes to oxidize the ⁇ boses of nucleic acids
- the recruitment linker is not nucleic acid, and instead may be any sort of linker or polymer As will be appreciated by those in the art generally any linker or polymer that can be modified to contain ETMs can be used
- a "recruitment polymer” comprises at least two or three subunits, which are covalently attached At least some portion of the monomenc subunits contain functional groups for the covalent attachment of ETMs
- coupling moieties are used to covalently link the subunits with the ETMs
- Preferred functional groups for attachment are ammo groups, carboxy groups, oxo groups and thiol groups, with ammo groups being particularly preferred
- Suitable linkers include, but are not limited to, alkyl linkers (including heteroalkyl (including (poly)ethylene glycol-type structures), substituted alkyl, aryalkyl linkers, etc As above for the polymers, the linkers will comprise one or more functional groups for the attachment of ETMs, which will be done as will be appreciated by those in the art, for example through the use homo-or hetero- bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference)
- Suitable recruitment polymers include, but are not limited to, functionahzed styrenes, such as ammo styrene, functionahzed dextrans, and polyamino acids
- Preferred polymers are polyamino acids (both poly-D-amino acids and poly-L-amino acids), such as polylysine, and polymers containing lysme and other ammo acids being particularly preferred
- charged recruitment linkers are preferred, for example when non-charged target analytes are to be detected
- Other suitable polyamino acids are polyglutamic acid, polyaspartic acid, co-polymers of lysme and glutamic or aspartic acid, co-polymers of lysme with alanme, tyros e, phenylalanine, serine, tryptophan, and/or prohne
- the recruitment linker comprises a metallocene polymer, as is described above
- the attachment of the recruitment linkers to the first portion of the label probe will depend on the composition of the recruitment linker, as will be appreciated by those in the art
- the recruitment linker is nucleic acid
- it is generally formed during the synthesis of the first portion of the label probe, with incorporation of nucleosides containing ETMs as required
- the first portion of the label probe and the recruitment linker may be made separately, and then attached
- there may be an overlapping section of complementarity, forming a section of double stranded nucleic acid that can then be chemically crosshnked, for example by using psoralen as is known in the art
- attachment of the linker/polymer of the recruitment linker will be done generally using standard chemical techniques, such as will be appreciated by those in the art
- attachment can be similar to the attachment of insulators to
- recruitment linkers that are mixtures of nucleic acids and non-nucleic acids, either in a linear form (i e nucleic acid segments linked together with alkyl linkers) or in branched forms (nucleic acids with alkyl "branches" that may contain ETMs and may be additionally branched) /
- the target analyte is a nucleic acid
- it is the target sequence itself that carries the ETMs, rather than the recruitment linker of a label probe
- t ⁇ phosphate nucleotides comprising the ETMs of the invention
- PCR polymerase chain reaction
- PCR amplification of a target sequence will result in target sequences comprising ETMs, generally randomly incorporated into the sequence
- ETMs ETMs
- compositions utilizing electrodes comprising monolayers with capture probes, and target sequences that comprises a first portion that is capable of hybridizing to a component of an assay complex, and a second portion that does not hybridize to a component of an assay complex and comprises at least one covalently attached electron transfer moiety Similarly, methods utilizing these compositions are also provided
- ETMs may be added to non-recruitment linkers as well
- ETMs may be added to sections of label probes that do hybridize to components of the assay complex, for example the first portion, or to the target sequence as outlined above
- These ETMs may be used for electron transfer detection in some embodiments, or they may not, depending on the location and system
- the target sequence containing randomly incorporated ETMs is hybridized directly to the capture probe, there may be ETMs in the portion hybridizing to the capture probe If the capture probe is attached to the electrode using a conductive oligomer, these ETMs can be used to detect electron transfer as has been previously described Alternatively, these ETMs may not be specifically detected
- the recruitment linker when the recruitment linker is nucleic acid, it may be desirable in some instances to have some or all of the recruitment linker be double stranded, for example in the mechan ⁇ sm-2 systems
- the first recruitment linker comprises the covalently attached ETMs
- the second recruitment linker contains the ETMs, and the first recruitment linker does not, and the ETMs are recruited to the surface by hybridization of the second recruitment linker to the first
- both the first and second recruitment linkers comprise ETMs It should be noted, as discussed above, that nucleic acids comprising a large number of ETMs may not hybridize as well, i e the T m may be decreased, depending on the site of attachment and the characteristics of the ETM Thus, in general, when multiple ETMs are used on hybridizing strands, i e in mechan ⁇ s
- the probes of the present invention are designed to be complementary to a target sequence (either the target sequence of the sample or to other probe sequences, as is described below), such that hybridization of the target sequence and the probes of the present invention occurs
- a target sequence either the target sequence of the sample or to other probe sequences, as is described below
- this complementarity need not be perfect, there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention
- the sequence is not a complementary target sequence
- substantially complementary herein is meant that the probes are sufficiently complementary to the target sequences to hybridize under normal reaction conditions
- nucleic acid compositions of the invention are useful as ohgonucleotide probes
- the length of the probe will vary with the length of the target sequence and the hybridization and wash conditions
- ohgonucleotide probes range from about 8 to about 50 nucleotides, with from about 10 to about 30 being preferred and from about 12 to about 25 being especially preferred
- very long probes may be used, e g 50 to 200-300 nucleotides in length
- nucleosides may be replaced with nucleic acids
- a variety of hybridization conditions may be used in the present invention, including high, moderate and low stringency conditions, see for example Maniatis et al , Molecular Cloning A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed Ausubel, et al, hereby incorporated by reference Stringent conditions are sequence-dependent and will be different in different circumstances Longer sequences hybridize specifically
- less stringent hybridization conditions are used, for example, moderate or low stringency conditions may be used, as are known in the art, see Maniatis and Ausubel, supra, and Tijssen, supra
- hybridization conditions may also vary when a non-ionic backbone, i e PNA is used, as is known in the art
- cross-linking agents may be added after target binding to cross-link, i e covalently attach the two strands of the hybridization complex
- the systems of the invention may take on a large number of different configurations, some of which are depicted in Figures 6 and 7
- there are three types of systems that can be used (1 ) systems in which the target sequence itself is labeled with ETMs (see Figure 6A, this is generally useful for nucleic acid systems), (2) systems in which label probes directly bind to the target analytes (see Figure 6C), and (3) systems in which label probes are indirectly bound to the target sequences, for example through the use of amplifier probes (see Figure 6E)
- the target sequence be immobilized on the electrode surface This is preferably done using capture probes and optionally one or more capture extender probes, see Figure 3 for representative nucleic acid examples
- capture extender probes may be used, that allow a universal ' surface, i e a surface containing a single type of capture probe that can be used to detect any target sequence
- Capture extender' probes are generally depicted in Figure 6C and other, and have a first portion that will hybridize to all or part of the capture probe, and a second portion that will hybridize to a portion of the target sequence This then allows the generation of customized soluble probes which as will be appreciated by those in the art is generally simpler and less costly
- two capture extender probes may be used This has generally been done to stabilize assay complexes (for example when the target sequence
- nucleic acid 'tail can be added to a binding ligand
- the binding ligands are added after the formation of the SAM ((4) above) This may be done in a variety of ways, as will be appreciated by those in the art
- conductive oligomers with terminal functional groups are made, with preferred embodiments utilizing activated carboxylates and isothiocyanaf.es, that will react with primary amines that are either present or put onto the binding ligand such as a nucleic acid, using an activated carboxylate
- activated carboxylates and isothiocyanaf.es
- SAMs comprising metal chelates is known, see U S Patent No 5,620,850 This allows the spotting of probes (either capture or detection probes, or both) using known methods (ink jet, spotting, etc ) onto the surface
- binding partner pairs can be utilized, i e one binding partner is attached to the terminus of the conductive oligomer, and the other to the end of the nucleic acid This may also be done without using a nucleic acid capture probe, that is, one binding partner serves as the capture probe and the other is attached to either the target sequence or a capture extender probe That is, either the target sequence comprises the binding partner, or a capture extender probe that will hybridize to the target sequence comprises the binding partner Suitable binding partner pairs include, but are not limited to, hapten pairs such as biotin/streptavidm, antigens/antibodies, NTA/histidme tags, etc In general, smaller binding partners are preferred, such that the electrons can pass from the nucleic acid into the conductive oligomer to allow detection
- the binding partner when the target sequence itself is modified to contain a binding partner, the binding partner is attached via a modified nucleotide that can be enzymatically attached to the target sequence, for example during a PCR target amplification step Alternatively, the binding partner should be easily attached to the target sequence
- a capture extender probe may be utilized that has a nucleic acid portion for hybridization to the target as well as a binding partner (for example, the capture extender probe may comprise a non-nucleic acid portion such as an alkyl linker that is used to attach a binding partner)
- the capture extender probe may comprise a non-nucleic acid portion such as an alkyl linker that is used to attach a binding partner
- the target sequence itself contains the ETMs
- this may be done using target sequences that have ETMs incorporated at any number of positions, as outlined above
- the 3'-5' orientation of the probes and targets is chosen to get the ETM-containmg structures (i e recruitment linkers or target sequences) as close to the surface of the monolayer as possible, and in the correct orientation This may be done using attachment via insulators or conductive oligomers as is generally shown in the Figures
- multiple capture probes can be utilized, either in a configuration such as depicted in Figure 6D, wherein the 5'-3' orientation of the capture probes is different, or where "loops" of target form when multiples of capture probes are used.
- the label probes directly hybridize to the target sequences, as is generally depicted in the figures
- the target sequence is immobilized on the surface using capture probes, including capture extender probes
- Label probes are then used to bring the ETMs into proximity of the surface of the monolayer
- multiple label probes are used, that is, label probes are designed such that the portion that hybridizes to the target sequence can be different for a number of different label probes, such that amplification of the signal occurs, since multiple label probes can bind for every target sequence
- n is an integer of at least one Depending on the sensitivity desired, the length of the target sequence, the number of ETMs per label probe, etc , preferred ranges of n are from 1 to 50, with from about 1 to about 20 being particularly preferred, and from about 2 to about 5 being especially preferred.
- label extender probes can be used as generally described below for use with
- the configuration of the system and the label probes are designed to recruit the ETMs as close as possible to the monolayer surface
- the label probes are hybridized to the target sequence indirectly That is, the present invention finds use in novel combinations of signal amplification technologies and electron transfer detection on electrodes, which may be particularly useful in sandwich hybridization assays, as generally depicted in the Figures for nucleic acid embodiments, similar systems can be developed for non-nucleic acid target analytes
- the amplifier probes of the invention are bound to the target sequence in a sample either directly or indirectly Since the amplifier probes preferably contain a relatively large number of amplification sequences that are available for binding of label probes, the detectable signal is significantly increased, and allows the detection limits of the target to be significantly improved
- These label and amplifier probes, and the detection methods described herein may be used in essentially any known nucleic acid hybridization formats, such as those in which the target is bound directly to a solid phase or in sandwich hybridization assays in which the target is bound to one or more nucleic acids that are in turn bound to the solid phase
- an amplifier probe is hybridized to the target sequence, either directly or through the use of a label extender probe which serves to allow ' generic' amplifier probes to be made
- the target sequence is preferably, but not required to be, immobilized on the electrode using capture probes
- the amplifier probe contains a multiplicity of amplification sequences, although in some embodiments, as described below, the amplifier probe may contain only a single amplification sequence
- the amplifier probe may take on a number of different forms, either a branched conformation, a dend ⁇ mer conformation, or a linear "string" of amplification sequences These amplification sequences are used to form hybridization complexes with label probes, and the ETMs can be detected using the electrode
- the present invention provides assay complexes comprising at least one amplifier probe
- amplifier probe or “nucleic acid multimer” or “amplification multimer” or grammatical equivalents herein is meant a nucleic acid probe that is used to facilitate signal amplification
- Amplifier probes comprise at least a first single-stranded nucleic acid probe sequence, as defined below, and at least one single-stranded nucleic acid amplification sequence, with a multiplicity of amplification sequences being preferred
- Amplifier probes comprise a first probe sequence that is used, either directly or indirectly, to hybridize to the target sequence That is, the amplifier probe itself may have a first probe sequence that is substantially complementary to the target sequence or it has a first probe sequence that is substantially complementary to a portion of an additional probe, in this case called a label extender probe, that has a first portion that is substantially complementary to the target sequence In a preferred embodiment, the first probe sequence of the amplifier probe is substantially complementary to the target sequence
- the first probe sequence is of a length sufficient to give specificity and stability
- the probe sequences of the invention that are designed to hybridize to another nucleic acid are at least about 5 nucleosides long, with at least about 10 being preferred and at least about 15 being especially preferred
- the amplifier probes, or any of the other probes of the invention may form hairpin stem-loop structures in the absence of their target The length of the stem double-stranded sequence will be selected such that the hairpin structure is not favored in the presence of target.
- these hairpin structures comprise four components
- the first component is a target binding sequence, i e a region complementary to the target (which may be the sample target sequence or another probe sequence to which binding is desired), that is about 10 nucleosides long, with about 15 being preferred
- the second component is a loop sequence, that can facilitate the formation of nucleic acid loops Particularly preferred in this regard are repeats of GTC, which has been identified in Fragile X Syndrome as forming turns (When PNA analogs are used, turns comprising prolme residues may be preferred) Generally, from three to five repeats are used, with four to five being preferred
- the third component is a self-complementary region, which has a first portion that is complementary to a portion of the target sequence region and a second portion that comprises a first portion of the label probe binding sequence
- the fourth component is substantially complementary to a label probe (or other probe, as the case may be)
- the fourth component further comprises a "sticky end", that is, a portion that does not hybridize to
- the amplifier probe provides an amplification of signal due to a multiplicity of labelling events, and several different amplifier probes, each with this multiplicity of labels, for each target sequence is used
- preferred embodiments utilize at least two different pools of amplifier probes, each pool having a different probe sequence for hybridization to different portions of the target sequence, the only real limitation on the number of different amplifier probes will be the length of the original target sequence
- the different amplifier probes contain different amplification sequences, although this is generally not preferred
- the amplifier probe does not hybridize to the sample target sequence directly, but instead hybridizes to a first portion of a label extender probe
- a label extender probe This is particularly useful to allow the use of ' generic' amplifier probes, that is, amplifier probes that can be used with a variety of different targets This may be desirable since several of the amplifier probes require special synthesis techniques
- the addition of a relatively short probe as a label extender probe is preferred
- the first probe sequence of the amplifier probe is substantially complementary to a first portion or domain of a first label extender single-stranded nucleic acid probe
- the label extender probe also contains a second portion or domain that is substantially complementary to a portion of the target sequence Both of these portions are preferably at least about 10 to about 50 nucleotides in length, with a range of about 15 to about 30 being preferred
- the terms "first" and "second” are not meant to confer an orientation of the sequences with respect to the 5'-3' orientation of the target or probe sequences For example, assuming a 5'-3
- more than one label extender probe-amplifier probe pair may be used, tht is, n is more than 1 That is, a plurality of label extender probes may be used, each with a portion that is substantially complementary to a different portion of the target sequence, this can serve as another level of amplification
- a preferred embodiment utilizes pools of at least two label extender probes, with the upper limit being set by the length of the target sequence
- more than one label extender probe is used with a single amplifier probe to reduce non-specific binding, as is generally outlined in U S. Patent No. 5,681 ,697, incorporated by reference herein
- a first portion of the first label extender probe hybridizes to a first portion of the target sequence
- the second portion of the first label extender probe hybridizes to a first probe sequence of the amplifier probe
- a first portion of the second label extender probe hybridizes to a second portion of the target sequence
- the second portion of the second label extender probe hybridizes to a second probe sequence of the amplifier probe
- the label extender probes may interact with a preamplifier probe, described below, rather than the amplifier probe directly
- a preferred embodiment utilizes several different amplifier probes, each with first probe sequences that will hybridize to a different portion of the label extender probe.
- the different amplifier probes contain different amplification sequences, although this is generally not preferred.
- the amplifier probe also comprises at least one amplification sequence
- An ' amplification sequence" or “amplification segment” or grammatical equivalents herein is meant a sequence that is used, either directly or indirectly, to bind to a first portion of a label probe as is more fully described below
- the amplifier probe comprises a multiplicity of amplification sequences, with from about 3 to about 1000 being preferred, from about 10 to about 100 being particularly preferred, and about 50 being especially preferred in some cases, for example when linear amplifier probes are used, from 1 to about 20 is preferred with from about 5 to about 10 being particularly preferred
- the amplification sequences may be linked to each other in a variety of ways, as will be appreciated by those in the art They may be covalently linked directly to each other, or to intervening sequences or chemical moieties, through nucleic acid linkages such as phosphodiester bonds, PNA bonds, etc , or through interposed linking agents such ammo acid, carbohydrate or polyol bridges, or through other cross-linking agents or binding partners
- the s ⁇ te(s) of linkage may be at the ends of a segment, and/or at one or more internal nucleotides in the strand
- the amplification sequences are attached via nucleic acid linkages
- branched amplifier probes are used, as are generally described in U S
- Branched amplifier probes may take on "forklike or comb-like' conformations 'Fork-like' branched amplifier probes generally have three or more ohgonucleotide segments emanating from a point of origin to form a branched structure
- the point of origin may be another nucleotide segment or a multifunctional molecule to whcih at least three segments can be covalently or tightly bound
- "Comb-like" branched amplifier probes have a linear backbone with a multiplicity of sidecham ohgonucleotides extending from the backbone
- the pendant segments will normally depend from a modified nucleotide or other organic moiety having the appropriate functional groups for attachment of ohgonucleotides
- a large number of amplification sequences are available for binding, either directly or indirectly, to detection probes
- these structures are made as is known in the art, using modified multifunctional nucleotides, as is
- dend ⁇ mer amplifier probes are used, as are generally described in U S Patent No 5,175,270, hereby expressly incorporated by reference
- Dend ⁇ me ⁇ c amplifier probes have amplification sequences that are attached via hybridization, and thus have portions of double-stranded nucleic acid as a component of their structure
- the outer surface of the dend ⁇ mer amplifier probe has a multiplicity of amplification sequences
- linear amplifier probes are used, that have individual amplification sequences linked end-to-end either directly or with short intervening sequences to form a polymer
- the linear amplifier probe has a single amplification sequence This may be useful when cycles of hyb ⁇ dization/disassociation occurs, forming a pool of amplifier probe that was hybridized to the target and then removed to allow more probes to bind, or when large numbers of ETMs are used for each label probe
- linear amplifier probes comprise a multiplicity of amplification sequences
- the amplifier probe may be totally linear totally branched, totally dend ⁇ me ⁇ c, or any combination thereof
- the amplification sequences of the amplifier probe are used, either directly or indirectly, to bind to a label probe to allow detection
- the amplification sequences of the amplifier probe are substantially complementary to a first portion of a label probe
- amplifier extender probes are used, that have a first portion that binds to the amplification sequence and a second portion that binds to the first portion of the label probe
- compositions of the invention may include "preamplifier” molecules, which serves a bridging moiety between the label extender molecules and the amplifier probes In this way, more amplifier and thus more ETMs are ultimately bound to the detection probes
- Preamplifier molecules may be either linear or branched, and typically contain in the range of about 30-3000 nucleotides
- tissue collection devices of the invention may be used in a variety of ways Components of the reaction may be added simultaneously, or sequentially, in any order, with preferred embodiments outlined below
- the invention thus provides assay complexes that generally minimally comprise a target sequence, a capture probe and a label probe (or in the case where the target analyte is labeled with the ETMs, the assay complex comprises a target sequence and a capture probe)
- Assay complex herein is meant the collection of attachment or hybridization complexes comprising analytes, including binding ligands and targets and at least one ETM, that allows detection
- the composition of the assay complex depends on the use of the different probe component outlined herein
- the assay complex comprises the capture probe and the target sequence
- a preferred embodiment utilizes a capture probe, a target sequence and a label probe
- the assay complexes may also include capture extender probes, label extender probes, and amplifier probes, as outlined herein, depending on the configuration used
- the assays are generally run under st ⁇ ngency conditions which allows formation of the label probe attachment complex only in the presence of target Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc
- Stringency may also include the use of an electrophoretic step to drive non-specific (i e low stringency) materials away from the detection electrode These parameters may also be used to control non-specific binding, as is generally outlined in U.S Patent No 5,681 ,697 Thus it may be desirable to perform certain steps at higher stringency conditions, for example, when an initial hybridization step is done between the target sequence and the label extender and capture extender probes Running this step at conditions which favor specific binding can allow the reduction of non-specific binding
- a preferred method is as follows Single-stranded target sequence is incubated under hybridization conditions with the capture extender probes and the label extender probes A preferred embodiment does this reaction in the presence of the electrode with immobilized capture probes, although this may also be done in two steps, with the initial incubation and the subsequent addition to the electrode
- Excess reagents are washed off, and amplifier probes are then added If preamplifier probes are used, they may be added either prior to the amplifier probes or simultaneously with the amplifier probes Excess reagents are washed off, and label probes are then added Excess reagents are washed off, and detection proceeds as outlined below
- a number of capture probes (or capture probes and capture extender probes that are each substantially complementary to a different portion of the target sequence are used
- the system when amplifier probes are used, the system is generally configured such that upon label probe binding, the recruitment linkers comprising the ETMs are placed in proximity either to. the monolayer surface containing conductive oligomers (mechan ⁇ sm-2) or in proximity to detection probes
- the length of the linkers from the nucleic acid point of attachment to the ETMs may vary, particularly with the length of the capture probe when capture extender probes are used That is, longer capture probes, with capture extenders, can result in the target sequences being "held” further away from the surface than for shorter capture probes Adding extra linking sequences between the probe nucleic acid and the ETMs can result in the ETMs being spatially closer to the surface, giving better results
- the length of the recruitment linker, the length of the detection probe, and their distance may be optimized.
- nucleic acids utilized in the invention may also be hgated together prior to detection, if applicable, by using standard molecular biology techniques such as the use of a gase Similarly, if desirable for stability, cross-linking agents may be added to hold the structures stable.
- compositions of the invention are generally synthesized as outlined below and in U S Patent Nos 5,591 ,578 5,824,473 5,770,369 5,705,348, 5,780,234, and 5,952,172 WO98/20162, W099/37819, PCT US98/12430, PCT US99/01703, PCT US98/12082, PCT/US99/14191 , PCT/US99/21683, PCT/US99/25464, PCT US99/10104, and U S S N s 09/135,183, 09/295,691 and 09/306,653, all of which are expressly incorporated by reference, and utilizing techniques well known in the art
- many of the techniques outlined below are directed to nucleic acids containing a nbose-phosphate backbone
- many alternate methods outlined below are directed to nucleic acids containing a nbose-phosphate backbone
- compositions may be made in several ways and will depend on the components as well as the mechanism used
- a preferred method first synthesizes an attachment linker attached to a nucleoside, with addition of additional nucleosides to form the capture probe followed by attachment to the electrode
- the whole capture probe may be made and then the completed attachment linker added, followed by attachment to the electrode
- a monolayer comprising attachment linkers (some of which have functional groups for attachment of capture probes) is attached to the electrode first, followed by attachment of the capture probe
- attachment linkers are used which are not stable in the solvents and under the conditions used in traditional nucleic acid synthesis
- compositions of the invention are made by first forming the attachment linker covalently attached to the nucleoside, followed by the addition of additional nucleosides to form a capture probe nucleic acid, with the last step comprising the addition of the attachment linker to the electrode
- attachment linker to the nucleoside may be done in several ways In a preferred embodiment, all or part of the attachment linker is synthesized first (generally with a functional group on the end for attachment to the electrode), which is then attached to the nucleoside Additional nucleosides are then added as required, with the last step generally being attachment to the electrode
- attachment linker is a conductive oligomer
- oligomer units are added one at a time to the nucleoside, with addition of additional nucleosides and attachment to the electrode
- a number of representative syntheses are shown in the Figures of WO 98/20162; PCT/US98/12430, PCT/US98/12082, PCT/US99/01705, PCT/US99/01703, and U S S N s 09/135,183, 60/105,875, and 09/295,691 , all of which are incorporated by reference
- the attachment linker is then attached to a nucleoside that may contain one (or more) of the oligomer units, attached as depicted herein
- attachment is to a ribose of the nbose-phosphate backbone, including amide and amine linkages
- attachment is via a phosphate of the nbose-phosphate backbone, as generally outlined in PCT US97/20014
- attachment is via the base
- protecting groups may be added to the base prior to addition of the conductive oligomers, as is generally known in the art
- palladium cross-coupling reactions may be altered to prevent dime ⁇ zation problems, i e two conductive oligomers dime ⁇ zing, rather than coupling to the base
- attachment to the base may be done by making the nucleoside with one unit of the, oligomer, followed by the addition of others
- modified nucleosides Once the modified nucleosides are prepared, protected and activated, prior to attachment to the electrode, they may be incorporated into a growing ohgonucleotide by standard synthetic techniques (Gait, Ohgonucleotide Synthesis A Practical Approach, IRL Press, Oxford, UK 1984, Eckstein) in several ways
- one or more modified nucleosides are converted to the t ⁇ phosphate form and incorporated into a growing ohgonucleotide chain by using standard molecular biology techniques such as with the use of the enzyme DNA polymerase I T4 DNA polymerase, T7 DNA polymerase, Taq DNA polymerase, reverse transcriptase, and RNA polymerases
- DNA polymerase I T4 DNA polymerase T4 DNA polymerase
- T7 DNA polymerase T7 DNA polymerase
- Taq DNA polymerase reverse transcriptase
- RNA polymerases for the incorporation of a 3' modified nucleoside to a nucleic acid, terminal deoxynucleotidyltransferase may be used (Rathff, Terminal deoxynucleotidyltransferase In The Enzymes, Vol 14A P D Boyer ed pp 105-118 Academic Press, San Diego, CA 1981 )
- the present invention provides deoxy ⁇ bonu
- nucleic acids comprising ETMs in situ
- a target sequence can hybridize to a capture probe (for example on the surface) in such a way that the terminus of the target sequence is exposed, i e unhyb ⁇ dized
- a capture probe for example on the surface
- enzyme and t ⁇ phosphate nucleotides labelled with ETMs allows the in situ creation of the label
- using labeled nucleotides recognized by polymerases can allow simultaneous PCR and detection, that is, the target sequences are generated in situ
- the modified nucleoside is converted to the phosphoramidite or H- phosphonate form, which are then used in solid-phase or solution syntheses of ohgonucleotides
- the modified nucleoside either for attachment at the ribose (i e ammo- or thiol-mod fled nucleosides) or the base, is incorporated into the ohgonucleotide at either an internal position or the 5' terminus
- DMT 4',4-d ⁇ methoxyt ⁇ tyl
- a preferred method utilizes the attachment of the modified nucleoside (or the nucleoside replacement) to controlled pore glass (CPG) or other ohgome ⁇ c supports
- CPG controlled pore glass
- the modified nucleoside is protected at the 5' end with DMT, and then reacted with succmic anhydride with activation
- the resulting succmyl compound is attached to CPG or other oligome ⁇ c supports as is known in the art
- Further phosphoramidite nucleosides are added, either modified or not, to the 5' end after deprotection
- the present invention provides conductive oligomers or insulators covalently attached to nucleosides attached to solid ohgome ⁇ c supports such as CPG, and phosphoramidite derivatives of the nucleosides of the invention
- the invention further provides methods of making label probes with recruitment linkers comprising ETMs
- ETMs these synthetic reactions will depend on the character of the recruitment linker and the method of attachment of the ETM, as will be appreciated by those in the art
- the label probes are generally made as outlined herein with the incorporation of ETMs at one or more positions
- a transition metal complex is used as the ETM
- synthesis may occur in several ways
- the l ⁇ gand(s) are added to a nucleoside, followed by the transition metal ion, and then the nucleoside with the transition metal complex attached is added to an ohgonucleotide, i e by addition to the nucleic acid synthesizer
- the l ⁇ gand(s) may be attached, followed by incorportation into a growing ohgonucleotide chain, followed by the addition of the metal ion
- ETMs are attached to a ribose of the nbose-phosphate backbone This is generally done as is outlined herein for conductive oligomers, as described herein, and in PCT publication WO 95/15971 , using amino-modified or oxo-modified nucleosides, at either the 2' or 3' position of the ribose
- the am o group may then be used either as a ligand, for example as a transition metal ligand for attachment of the metal ion, or as a chemically functional group that can be used for attachment of other ligands or organic ETMs, for example via amide linkages, as will be appreciated by those in the art
- the examples describe the synthesis of nucleosides with a variety of ETMs attached via the ribose
- ETMs are attached to a phosphate of the nbose-phosphate backbone As outlined herein, this may be done using phosphodiester analogs such as phosphoramidite bonds, see generally PCT publication WO 95/15971 , or can be done in a similar manner to that described in PCT US97/20014, where the conductive oligomer is replaced by a transition metal ligand or complex or an organic ETM
- ETMs are attached to a base of the nucleoside
- ammo groups of the base either naturally occurring or added as is described herein (see the fngures, for example) are used either as ligands for transition metal complexes or as a chemically functional group that can be used to add other ligands, for example via an amide linkage, or organic ETMs
- nucleosides containing halogen atoms attached to the heterocyclic ring are commercially available Acetylene linked ligands may be added using the halogenated bases, as is generally known, see for example, Tzahs et al , Tetrahedron Lett 36(34) 6017-6020 (1995) Tzahs et al , Tetrahedron Lett 36(2) 3489-3490 (1995), and Tzahs et al , Chem Communications (in press) 1996, all
- the nucleosides are made with transition metal ligands, incorporated into a nucleic acid, and then the transition metal ion and any remaining necessary ligands are added as is known in the art In an alternative embodiment, the transition metal ion and additional ligands are added prior to incorporation into the nucleic acid
- the attachment linker is attached to the electrode
- the method will vary depending on the type of electrode used As is described herein, the attachment linkers are generally made with a terminal "A' linker to facilitate attachment to the electrode
- a sulfur-gold attachment is considered a covalent attachment
- conductive oligomers, insulators, and attachment linkers are covalently attached via sulfur linkages to the electrode
- traditional protecting groups for use of attaching molecules to gold electrodes are generally not ideal for use in both synthesis of the compositions described herein and inclusion in ohgonucleotide synthetic reactions
- the present invention provides novel methods for the attachment of conductive oligomers to gold electrodes, utilizing unusual protecting groups, including ethylpyndme, and tnmethylsilylethyl as is depicted in the Figures
- unusual protecting groups including ethylpyndme, and tnmethylsilylethyl as is depicted in the Figures
- traditional protecting groups such as acetyl groups and others may be used See Greene et al , supra
- the subunit of the conductive oligomer which contains the sulfur atom for attachment to the electrode is protected with an ethyi-py ⁇ dine or tnmethylsilylethyl group
- this is generally done by contacting the subunit containing the sulfur atom (preferably in the form of a sulfhydryl) with a vinyl pyridine group or vinyl tnmethylsilylethyl group under conditions whereby an ethylpyndme group or tnmethylsilylethyl group is added to the sulfur atom
- This subunit also generally contains a functional moiety for attachment of additional subunits, and thus additional subunits are attached to form the conductive oligomer
- the conductive oligomer is then attached to a nucleoside, and additional nucleosides attached
- the protecting group is then removed and the sulfur-gold covalent attachment is made Alternatively, all or part of the conductive oligomer is made, and then either a subunit containing a protected sulfur atom is added, or a sulfur atom is added and then protected
- the conductive oligomer is then attached to a nucleoside, and additional nucleosides attached Alternatively, the conductive oligomer attached to a nucleic acid is made, and then either a subunit containing a protected sulfur atom is added, or a sulfur atom is added and then protected
- the ethyl pyridine protecting group may be used as above, but removed after one or more steps and replaced with a standard protecting group like a disulfide Thus, the ethy
- subunit of a conductive polymer herein is meant at least the moiety of the conductive oligomer to which the sulfur atom is attached, although additional atoms may be present, including either functional groups which allow the addition of additional components of the conductive oligomer, or additional components of the conductive oligomer
- a subunit comprises at least the first Y group
- a preferred method comprises 1 ) adding an ethyl pyridine or tnmethylsilylethyl protecting group to a sulfur atom attached to a first subunit of a conductive oligomer, generally done by adding a vinyl # pyridine or tnmethylsilylethyl group to a sulfhydryl, 2) adding additional subunits to form the conductive oligomer; 3) adding at least a first nucleoside to the conductive oligomer, 4) adding additional nucleosides to the first nucleoside to form a nucleic acid, 5) attaching the conductive oligomer to the gold electrode This may also be done in the absence of nucleosides, as is described in the Examples
- the above method may also be used to attach insulator molecules to a gold electrode
- a monolayer comprising surface species is added to the electrode.
- the chemistry of addition is similar to or the same as the addition of conductive oligomers to the electrode, i e using a sulfur atom for attachment to a gold electrode, etc
- Compositions comprising monolayers in addition to the conductive oligomers covalently attached to nucleic acids may be made in at least one of five ways (1 ) addition of the monolayer, followed by subsequent addition of the attachment hnker-nucleic acid complex, (2) addition of the attachment nker-nucleic acid complex followed by addition of the monolayer, (3) simultaneous addition of the monolayer and attachment hnker-nucleic acid complex, (4) formation of a monolayer (using any of 1 , 2 or 3) which includes attachment linkers which terminate in a functional moiety suitable for attachment of a completed nucleic acid, or (5) formation of a monolayer which includes attachment linkers which terminate in a functional moiety suitable
- the moieties can be attached either to a base or to the backbone of the monomenc subunit Attachment to the base is done as outlined herein or known in the literature
- the moieties are added to a base which is then incorporated into a PNA as outlined herein
- the base may be either protected, as required for incorporation into the PNA synthetic reaction, or de ⁇ vatized, to allow incorporation, either prior to the addition of the chemical substituent or afterwards Protection and de ⁇ vatization of the bases is shown in PCT US97/20014
- the bases can then be incorporated into monomenc subunits
- the moieties are covalently attached to the backbone of the PNA monomer
- the attachment is generally to one of the unsubstituted carbon atoms of the monomenc subunit, preferably the ⁇ -carbon of the backbone, although attachment at either of the carbon 1 or 2 positions, or the ⁇ -carbon of the amide bond linking the base to the backbone may be done
- other carbons or atoms may be substituted as well
- moieties are added at the ⁇ -carbon atoms, either to a terminal monomenc subunit or an internal one
- a modified monomenc subunit is synthesized with an ETM or an attachment linker, or a functional group for its attachment, and then the base is added and the modified monomer can be incorporated into a growing PNA chain
- compositions of the invention may additionally contain, in addition to ETMs, one or more other labels at any position
- label' herein is meant an element (e g an isotope) or chemical compound that is attached to enable the detection of the compound
- Preferred labels are radioactive isotopic labels, colored or fluorescent dyes and electrochemical labels such as ETMs
- the labels may be incorporated into the compound at any position
- the compositions of the invention may also contain other moieties such as cross-linking agents to facilitate cross-linking of the target-probe complex See for example, Lukhtanov et al , Nucl Acids Res 24(4) 683 (1996) and Tabone et al , Biochem 33 375 (1994), both of which are expressly incorporated by reference
- compositions find use in a number of applications, as described herein
- the compositions of the invention find use in binding assays for the detection of target analytes, in particular nucleic acid target sequences
- electrodes can be made that have a single species of binding ligand, or multiple binding ligand species, i e in an array format
- detection proceeds with electronic initiation Without being limited by the mechanism or theory, detection is based on the transfer of electrons from the ETM to the electrode, including via the " ⁇ -way"
- Detection of electron transfer, i e the presence of the ETMs is generally initiated electronically, with voltage being preferred
- a potential is applied to the assay complex
- Precise control and variations in the applied potential can be via a potentiostat and either a three electrode system (one reference, one sample (or working) and one counter electrode) or a two electrode system (one sample and one counter electrode)
- This allows matching of applied potential to peak potential of the system which depends in part on the choice of ETMs and in part on the conductive oligomer used, the composition and integrity of the monolayer, and what type of reference electrode is used
- ferrocene is a preferred ETM
- a co-reductant or co-oxidant (collectively, co-redoxant) is used, as an additional electron source or sink See generally Sato et al , Bull Chem Soc Jpn 66 1032 (1993), Uosaki et al , Electrochimica Acta 36 1799 (1991 ), and Alieman et al , J Phys Chem 100 17050
- an input electron source in solution is used in the initiation of electron transfer, preferably when initiation and detection are being done using DC current or at AC frequencies where diffusion is not limiting
- preferred embodiments utilize monolayers that contain a minimum of holes , such that short-circuiting of the system is avoided This may be done in several general ways
- an input electron source is used that has a lower or similar redox potential than the ETM of the label probe
- both the ETM and the input electron source are oxidized and can thus donate electrons, the ETM donates an electron to the electrode and the input source donates to the ETM
- ferrocene as a ETM attached to the compositions of the invention as described in the examples, has a redox potential of roughly 200 mV in aqueous solution (which can change significantly depending on what the ferrocene is bound to, the manner of the linkage and the
- input electron sources that have lower redox potentials than the ETM are used. At voltages less than the redox potential of the ETM, but higher than the redox potential of the electron source, the input source such as ferrocyanide is unable to be oxided and thus is unable to donate an electron to the ETM, i e no electron transfer occurs Once ferrocene is oxidized, then there is a pathway for electron transfer
- an input electron source is used that has a higher redox potential than the ETM of the label probe
- luminol an electron source
- the ETM is unable to accept any electrons from the luminol electron source, since the voltages are less than the redox potential of the luminol
- the luminol then transfers an electron to the ETM, allowing rapid and repeated electron transfer
- the electron source (or co- reductant) serves to amplify the signal generated in the system, as the electron source molecules rapidly and repeatedly donate electrons to the ETM of the label probe
- Luminol has the added benefit of becoming a chemiiummiscent species upon oxidation (see Jirka et al , Analytica Chimica Acta 284 345 (1993)), thus allowing photo-detection of electron transfer from the
- luminol to the electrode
- the luminol can only be oxidized by transferring an electron to the ETM on the label probe
- the ETM is not present, i e when the target sequence is not hyb ⁇ dized to the composition of the invention, luminol is not significantly oxidized, resulting in a low photon emission and thus a low (if any) signal from the luminol In the presence of the target, a much larger signal is generated
- the measure of luminol oxidation by photon emission is an indirect measurement of the ability of the ETM to donate electrons to the electrode
- photon detection is generally more sensitive than electronic detection, the sensitivity of the system may be increased
- Suitable electron source molecules are well known in the art, and include, but are not limited to, fer ⁇ cyanide, and luminol
- output electron acceptors or sinks could be used, i e the above reactions could be run in reverse, with the ETM such as a metallocene receiving an electron from the electrode, converting it to the metalhcenium, with the output electron acceptor then accepting the electron rapidly and repeatedly
- cobalticenium is the preferred ETM
- the presence of the ETMs at the surface of the monolayer can be detected in a variety of ways
- a variety of detection methods may be used, including, but not limited to, optical detection (as a result of spectral changes upon changes in redox states), which includes fluorescence, phosphorescence, luminiscence, chemiluminescence, electrochemilummescence, and refractive index; and electronic detection, including, but not limited to, amperommetry, voltammetry, capacitance and impedence
- optical detection as a result of spectral changes upon changes in redox states
- electronic detection including, but not limited to, amperommetry, voltammetry, capacitance and impedence
- These methods include time or frequency dependent methods based on AC or DC currents, pulsed methods, lock-in techniques, filtering (high pass, low pass, band pass), and time-resolved techniques including time-resolved fluoroscence
- the efficient transfer of electrons from the ETM to the electrode results in stereotyped changes in the redox state of the ETM
- ETMs including the complexes of ruthenium containing bipy ⁇ dine, pyridine and imidazole rings
- these changes in redox state are associated with changes in spectral properties
- Significant differences in absorbance are observed between reduced and oxidized states for these molecules See for example Fabbnzzi et al , Chem Soc Rev 1995 pp197-202) These differences can be monitored using a spectrophotometer or simple photomultipher tube device
- possible electron donors and acceptors include all the derivatives listed above for photoactivation or initiation Preferred electron donors and acceptors have characteristically large spectral changes upon oxidation and reduction resulting in highly sensitive monitoring of electron transfer Such examples include Ru(NH 3 ) 4 py and Ru(bpy) 2 ⁇ m as preferred examples It should be understood that only the donor or acceptor that is being monitored by absorbance need have ideal spectral characteristics
- the electron transfer is detected fluoromet ⁇ cally Numerous transition metal complexes, including those of ruthenium, have distinct fluorescence properties Therefore, the change in redox state of the electron donors and electron acceptors attached to the nucleic acid can be monitored very sensitively using fluorescence, for example with Ru(4,7-b ⁇ phenyl 2 -phenanthrol ⁇ ne) 3 2+
- fluorescence for example with Ru(4,7-b ⁇ phenyl 2 -phenanthrol ⁇ ne) 3 2+
- the production of this compound can be easily measured using standard fluorescence assay techniques For example, laser induced fluorescence can be recorded in a standard single cell fluo ⁇ meter, a flow through "on-line” fluo ⁇ meter (such as those attached to a chromatography system) or a multi-sample "plate-reader” similar to those marketed for 96-well immuno assays
- fluorescence can be measured using fiber optic sensors with nucleic acid probes in solution or attached to the fiber optic Fluorescence is monitored using a photomultipher tube or other light detection instrument attached to the fiber optic The advantage of this system is the extremely small volumes of sample that can be assayed
- scanning fluorescence detectors such as the Fluorlmager sold by Molecular Dynamics are ideally suited to monitoring the fluorescence of modified nucleic acid molecules arrayed on solid surfaces
- Fluorlmager sold by Molecular Dynamics
- transition metal complexes display fluorescence with large Stokes shifts Suitable examples include bis- and tnsphenanthrolme complexes and bis- and t ⁇ sbipy ⁇ dyl complexes of transition metals such as ruthenium (see Juris, A , Balzani, V , et al Coord Chem.
- Preferred examples display efficient fluorescence (reasonably high quantum yields) as well as low reorganization energies These include Ru(4,7-b ⁇ phenyl 2 -phenanthrohne) 3 2 ⁇ Ru(4,4'-d ⁇ phenyl-2,2'- b ⁇ pyr ⁇ d ⁇ ne) 3 2+ and platinum complexes (see Cummings et al , J Am Chem Soc 1 18 1949-1960 (1996), incorporated by reference) Alternatively, a reduction in fluorescence associated with hybridization can be measured using these systems
- electrochemilummescence is used as the basis of the electron transfer detection With some ETMs such as Ru 2+ (bpy) 3 , direct luminescence accompanies excited state decay Changes in this property are associated with nucleic acid hybridization and can be monitored with a simple photomultipher tube arrangement (see Blackburn, G F Clin Chem 37 1534-1539 (1991 ), and Juris et al , supra
- electronic detection is used, including amperommetry, voltammetry, capacitance, and impedence Suitable techniques include, but are not limited to, electrogravimetry, coulometry (including controlled potential coulometry and constant current coulometry), voltametry (cyclic voltametry, pulse voltametry (normal pulse voltametry, square wave voltametry, differential pulse voltametry, Osteryoung square wave voltametry, and coulostatic pulse techniques), stripping analysis
- monitoring electron transfer is via amperomet ⁇ c detection
- This method of detection involves applying a potential (as compared to a separate reference electrode) between the nucleic acid-conjugated electrode and a reference (counter) electrode in the sample containing target genes of interest Electron transfer of differing efficiencies is induced in samples in the presence or absence of target nucleic acid, that is, the presence or absence of the target nucleic acid, and thus the label probe, can result in different currents
- the device for measuring electron transfer amperomet ⁇ cally involves sensitive current detection and includes a means of controlling the voltage potential, usually a potentiostat This voltage is optimized with reference to the potential of the electron donating complex on the label probe Possible electron donating complexes include those previously mentioned with complexes of iron, osmium, platinum, cobalt, rhenium and ruthenium being preferred and complexes of iron being most preferred
- potentiomet ⁇ c or voltammet ⁇ c measurements involve non-faradaic (no net current flow) processes and are utilized traditionally in pH and other ion detectors Similar sensors are used to monitor electron transfer between the ETM and the electrode.
- other properties of insulators such as resistance
- conductors such as conductivity, impedance and capicitance
- any system that generates a current also generates a small magnetic field, which may be monitored in some embodiments
- Equation 1 Equation 1
- Equation 1 n is the number of electrons oxidized or reduced per redox molecule, f is the applied frequency, F is Faraday's constant, N tota: is the total number of redox molecules, E 0 is the formal potential of the redox molecule, R is the gas constant, T is the temperature in degrees Kelvin, and E DC is the electrode potential.
- the faradaic current can also be expressed as a function of time, as shown in Equation 2
- I f is the Faradaic current and q 0 is the elementary charge
- Equation 1 does not incorporate the effect of electron transfer rate nor of instrument factors
- Electron transfer rate is important when the rate is close to or lower than the applied frequency
- ⁇ AC f(Nernst factors )f(k ET )f( ⁇ nstrument factors)
- non-specifically bound label probes/ETMs show differences in impedance (i e higher impedances) than when the label probes containing the ETMs are specifically bound in the correct orientation
- the non-specifically bound material is washed away, resulting in an effective impedance of infinity
- AC detection gives several advantages as is generally discussed below, including an increase in sensitivity, and the ability to "filter out" background noise
- changes in impedance including, for example, bulk impedance
- frequency response when using AC initiation and detection methods, the frequency response of the system changes as a result of the presence of the ETM
- frequency response herein is meant a modification of signals as a result of electron transfer between the electrode and the ETM This modification is different depending on signal frequency
- a frequency response includes AC currents at one or more frequencies, phase shifts, DC offset voltages, faradaic impedance, etc
- a first input electrical signal is then applied to the system, preferably via at least the sample electrode (containing the complexes of the invention) and the counter electrode, to initiate electron transfer between the electrode and the ETM Three electrode systems may also be used, with the voltage applied to the reference and working electrodes
- the first input signal comprises at least an AC component
- the AC component may be of variable amplitude and frequency Generally, for use in the present methods, the AC amplitude ranges from about 1 mV to about 1 1 V, with from about 10 mV to about 800 mV being preferred, and from about 10 mV to about 500 mV being especially preferred
- the AC frequency ranges from about 0 01 Hz to about 100 MHz, with from about 10 Hz to about 10 MHz being preferred, and from about 100 Hz to about 20 MHz being especially preferred
- the first input signal comprises a DC component and an AC component
- a DC offset voltage between the sample and counter electrodes is swept through the electrochemical potential of the ETM (for example, when ferrocene is used, the sweep is generally from 0 to 500 mV) (or alternatively, the working electrode is grounded and the reference electrode is swept from 0 to -500 mV)
- the sweep is used to identify the DC voltage at which the maximum response of the system is seen This is generally at or about the electrochemical potential of the ETM
- either a sweep or one or more uniform DC offset voltages may be used DC offset voltages of from about -1 V to about +1 1 V are preferred, with from about -500 mV to about +800 mV being especially preferred, and from about -300 mV to about 500 mV being particularly preferred
- the DC offset voltage is not zero
- an AC signal component of variable amplitude and frequency is applied If the ETM is present, and can respond to the AC perturbation, an AC current will be produced due to electron transfer between the
- a single input signal may be applied to differentiate between the presence and absence of the ETM (i e the presence of the target sequence) nucleic acid
- a plurality of input signals are applied as outlined herein, this may take a variety of forms, including using multiple frequencies, multiple DC offset voltages, or multiple AC amplitudes, or combinations of any or all of these
- DC offset voltages are used, although as outlined above, DC voltage sweeps are preferred This may be done at a single frequency, or at two or more frequencies
- the AC amplitude is varied Without being bound by theory, it appears that increasing the amplitude increases the driving force Thus, higher amplitudes, which result in higher overpotentials give faster rates of electron transfer Thus, generally, the same system gives an improved response (i e higher output signals) at any single frequency through the use of higher overpotentials at that frequency Thus, the amplitude may be increased at high frequencies to increase the rate of electron transfer through the system, resulting in greater sensitivity In addition, this may be used, for example, to induce responses in slower systems such as those that do not possess optimal spacing configurations
- measurements of the system are taken at at least two separate amplitudes or overpotentials, with measurements at a plurality of amplitudes being preferred.
- changes in response as a result of changes in amplitude may form the basis of identification, calibration and quantification of the system
- one or more AC frequencies can be used as well
- the AC frequency is varied At different frequencies, different molecules respond in different ways As will be appreciated by those in the art, increasing the frequency generally increases the output current However, when the frequency is greater than the rate at which electrons may travel between the electrode and the ETM, higher frequencies result in a loss or decrease of output signal At some point, the frequency will be greater than the rate of electron transfer between the ETM and the electrode and then the output signal will also drop
- detection utilizes a single measurement of output signal at a single frequency That is, the frequency response of the system in the absence of target sequence, and thus the absence of label probe containing ETMs, can be previously determined to be very low at a particular high frequency Using this information, any response at a particular frequency, will show the presence of the assay complex That is, any response at a particular frequency is characteristic of the assay complex Thus it may only be necessary to use a single input high frequency, and any changes in frequency response is an indication that the ETM is present, and thus that the target sequence is present
- measurements of the system are taken at at least two separate frequencies, with measurements at a plurality of frequencies being preferred
- a plurality of frequencies includes a scan For example, measuring the output signal, e g , the AC current, at a low input frequency such as 1 - 20 Hz, and comparing the response to the output signal at high frequency such as 10 - 100 kHz will show a frequency response difference between the presence and absence of the ETM
- the frequency response is determined at at least two, preferably at least about five, and more preferably at least about ten frequencies
- an output signal After transmitting the input signal to initiate electron transfer, an output signal is received or detected
- the presence and magnitude of the output signal will depend on a number of factors, including the overpotential/amplitude of the input signal, the frequency of the input AC signal, the composition of the intervening medium, the DC offset, the environment of the system, the nature of the ETM, the solvent, and the type and concentration of salt
- the presence and magnitude of the output signal will depend in general on the presence or absence of the ETM, the placement and distance of the ETM from the surface of the monolayer and the character of the input signal
- the output signal comprises an AC current
- the magnitude of the output current will depend on a number of parameters By varying these parameters, the system may be optimized in a number of ways
- AC currents generated in the present invention range from about 1 femptoamp to about 1 milhamp, with currents from about 50 femptoamps to about 100 microamps being preferred, and from about 1 picoamp to about 1 microamp being especially preferred
- the output signal is phase shifted in the AC component relative to the input signal
- the systems of the present invention may be sufficiently uniform to allow phase-shifting based detection That is, the complex biomolecules of the invention through which electron transfer occurs react to the AC input in a homogeneous manner, similar to standard electronic components, such that a phase shift can be determined This may serve as the basis of detection between the presence and absence of the ETM, and/or differences between the presence of target-specific assay complexes comprising label probes and non-specific binding of the label probes to the system components
- the output signal is characteristic of the presence of the ETM, that is, the output signal is characteristic of the presence of the target-specific assay complex comprising label probes and ETMs
- the basis of the detection is a difference in the faradaic impedance of the system as a result of the formation of the assay complex Faradaic impedance is the impedance of the system between the electrode and the ETM Faradaic impedance is quite different from the bulk or dielectric impedance, which is the impedance of the bulk solution between the electrodes Many factors may change the faradaic impedance which may not effect the bulk impedance, and vice versa
- the assay complexes comprising the nucleic acids in this system have a certain faradaic impedance, that will depend on the distance between the ETM and the electrode, their electronic properties, and the composition of the intervening medium, among other things Of importance in the methods of the invention is that the faradaic impedance between the ETM and the electrode is signficantly different depending on whether the
- the present invention further provides electronic devices or apparatus for the detection of analytes using the compositions of the invention
- the apparatus includes a test chamber for receiving a sample solution which has at least a first measuring or sample electrode, and a second measuring or counter electrode
- a test chamber for receiving a sample solution which has at least a first measuring or sample electrode, and a second measuring or counter electrode
- the first and second measuring electrodes are in contact with a test sample receiving region, such that in the presence of a liquid test sample, the two electrophoresis electrodes may be in electrical contact
- the apparatus also includes detection electrodes comprising a single stranded nucleic acid capture probe covalently attached via an attachment linker, and a monolayer comprising conductive oligomers, such as are described herein
- the apparatus further comprises an AC voltage source electrically connected to the test chamber, that is, to the measuring electrodes
- the AC voltage source is capable of delivering DC offset voltage as well
- the apparatus further comprises a processor capable of comparing the input signal and the output signal
- the processor is coupled to the electrodes and configured to receive an output signal, and thus detect the presence of the target nucleic acid
- compositions of the present invention may be used in a variety of research, clinical, quality control, or field testing settings as outlined herein
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- Pathology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Sampling And Sample Adjustment (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
Abstract
La présente invention concerne des dispositifs de prélèvement de tissu, en particulier des dispositifs de prélèvement de sang, qui comportent une électrode. Les électrodes peuvent en outre comporter des monocouches auto-assemblées et des ligands de capture par liaison, notamment des sondes de capture d'acides nucléiques. Les monocouches peuvent comporter des espèces chimiques isolantes et/ou électroconductrices. Les dispositifs peuvent en outre comporter au moins un réactif, y compris des anticoagulants, des acides nucléiques sondes, et des réactifs de lyse. Dans un autre aspect, l'invention concerne un procédé de détection d'un analyte voulu dans un échantillon, qui comporte les étapes consistant à appliquer un signal d'initiation à un dispositif de prélèvement de tissu comportant une électrode. L'électrode peut comporter une monocouche auto-assemblée et un complexe de titrage contenant un ligand de capture par liaison, l'analyte voulu et une fraction de transfert d'électrons. Les procédés comportent en outre une détection de transfert d'électrons entre l'électrode et la fraction de transfert d'électrons. Les procédés peuvent en outre comporter une étape de prélèvement d'échantillon, par exemple à l'aide de l'équipement de prélèvement de sang.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11417898P | 1998-12-30 | 1998-12-30 | |
US114178P | 1998-12-30 | ||
PCT/US1999/031051 WO2000038836A1 (fr) | 1998-12-30 | 1999-12-27 | Dispositifs de prelevement de tissu contenant des biocapteurs |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1140360A1 true EP1140360A1 (fr) | 2001-10-10 |
Family
ID=22353771
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP99965340A Withdrawn EP1140360A1 (fr) | 1998-12-30 | 1999-12-27 | Dispositifs de prelevement de tissu contenant des biocapteurs |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1140360A1 (fr) |
JP (1) | JP2002533694A (fr) |
AU (1) | AU3128200A (fr) |
CA (1) | CA2355875A1 (fr) |
WO (1) | WO2000038836A1 (fr) |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2002513916A (ja) * | 1998-05-06 | 2002-05-14 | クリニカル・マイクロ・センサーズ・インコーポレイテッド | 単層を利用する被検体の電子的検出方法 |
US6761816B1 (en) | 1998-06-23 | 2004-07-13 | Clinical Micro Systems, Inc. | Printed circuit boards with monolayers and capture ligands |
AU1241000A (en) | 1998-10-27 | 2000-05-15 | Clinical Micro Sensors, Inc. | Detection of target analytes using particles and electrodes |
KR20050008693A (ko) * | 2002-04-22 | 2005-01-21 | 호쿠토카가쿠산교 카부시키사이샤 | 유전자 검출용구, 검출방법 및 검출용 키트 |
CN1860366A (zh) * | 2003-09-29 | 2006-11-08 | 皇家飞利浦电子股份有限公司 | 免标记检测生物分子 |
JP5214723B2 (ja) * | 2007-04-30 | 2013-06-19 | ナノゲン・インコーポレイテッド | 多分析物アッセイ |
WO2014066704A1 (fr) | 2012-10-24 | 2014-05-01 | Genmark Diagnostics, Inc. | Analyse cible à multiplexe intégré |
US10823699B2 (en) | 2016-07-29 | 2020-11-03 | Vital Biosciences, Inc. | Gel electrophoresis diagnostic kit and methods of using the same |
JP6876911B2 (ja) * | 2017-02-21 | 2021-05-26 | ソニーグループ株式会社 | 電気的測定用カートリッジ、電気的測定装置及び電気的測定方法 |
CN211955507U (zh) * | 2019-02-25 | 2020-11-17 | 上海快灵生物科技有限公司 | 一种生化反应试纸条管及试剂盒 |
CN113624823B (zh) * | 2021-08-04 | 2023-06-06 | 中国科学院上海高等研究院 | 基于四面体纳米结构dna的信号探针、其制备方法和用途 |
CN117417819A (zh) * | 2022-11-03 | 2024-01-19 | 深圳先进技术研究院 | 一种集成全封闭式核酸检测***以及检测装置 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05236997A (ja) * | 1992-02-28 | 1993-09-17 | Hitachi Ltd | ポリヌクレオチド捕捉用チップ |
US5824473A (en) * | 1993-12-10 | 1998-10-20 | California Institute Of Technology | Nucleic acid mediated electron transfer |
US5603711A (en) * | 1995-01-20 | 1997-02-18 | Everest Medical Corp. | Endoscopic bipolar biopsy forceps |
CA2270633A1 (fr) * | 1996-11-05 | 1998-05-14 | Clinical Micro Sensors | Electrodes reliees par l'intermediaire d'oligomeres conducteurs a des acides nucleiques |
ATE498838T1 (de) * | 1997-06-12 | 2011-03-15 | Clinical Micro Sensors Inc | Elektronische verfahren und vorrichtung zum nachweis von analyten |
DE19725190A1 (de) * | 1997-06-14 | 1998-12-17 | Innova Gmbh | Vorrichtungen mit integrierten Elektroden aus elektrisch leitfähigen Kunststoffen |
-
1999
- 1999-12-27 CA CA002355875A patent/CA2355875A1/fr not_active Abandoned
- 1999-12-27 AU AU31282/00A patent/AU3128200A/en not_active Abandoned
- 1999-12-27 WO PCT/US1999/031051 patent/WO2000038836A1/fr not_active Application Discontinuation
- 1999-12-27 EP EP99965340A patent/EP1140360A1/fr not_active Withdrawn
- 1999-12-27 JP JP2000590780A patent/JP2002533694A/ja active Pending
Non-Patent Citations (1)
Title |
---|
See references of WO0038836A1 * |
Also Published As
Publication number | Publication date |
---|---|
CA2355875A1 (fr) | 2000-07-06 |
AU3128200A (en) | 2000-07-31 |
WO2000038836A1 (fr) | 2000-07-06 |
JP2002533694A (ja) | 2002-10-08 |
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